Human cDNAs encoding polypeptides having kinase functions

ABSTRACT

The invention is directed to purified and isolated human polypeptides having kinase function, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, the use of such polypeptides and fragmented peptides in phosphorylation reactions and as molecular weight markers, the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, the use of such polypeptides in screening assays, and kits comprising these reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national application under 35 U.S.C. § 371 ofInternational Application No. PCT/US99/17630, having an internationalfiling date of Aug. 3, 1999; which claims the priority of provisionalapplications U.S. Ser. No. 60/095,270, filed Aug. 4, 1998, and U.S. Ser.No. 60/099,972, filed Sep. 11, 1998; all of which are incorporated byreference herein.

FIELD OF THE INVENTION

The invention is directed to purified and isolated human polypeptideshaving kinase function, the nucleic acids encoding such polypeptides,processes for production of recombinant forms of such polypeptides,antibodies generated against these polypeptides, fragmented peptidesderived from these polypeptides, the use of such polypeptides andfragmented peptides in phosphorylation reactions and as molecular weightmarkers, the use of such polypeptides and fragmented peptides ascontrols for peptide fragmentation, the use of such polypeptides inscreening assays, and kits comprising these reagents.

BACKGROUND OF THE INVENTION

The eukaryotic protein kinases make up a large and rapidly expandingfamily of proteins related on the basis of homologous catalytic domains.Spurred by the development of gene cloning and sequencing methodologies,distinct protein kinase genes have been identified from a wide selectionof invertebrates and lower eukaryotes, including Drosophila,Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding(Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe)yeast. Homologous genes have also been identified in higher plants.Protein kinases, however, are not limited to the eukaryotes. Enzymeactivities have been well documented in prokaryotes, but the prokaryoticprotein kinase genes are not obviously homologous to those of theeukaryotes. Because protein kinases are useful biochemical reagents,there is a need in the art for the continued discovery of unique membersof the protein kinase family.

In addition, the discovery and identification of proteins are at theforefront of modern molecular biology and biochemistry. Theidentification of the primary structure, or sequence, of a sampleprotein is the culmination of an arduous process of experimentation. Inorder to identify an unknown sample protein, the investigator can relyupon comparison of the unknown sample protein to known peptides using avariety of techniques known to those skilled in the art. For instance,proteins are routinely analyzed using techniques such aselectrophoresis, sedimentation, chromatography, and mass spectrometry.

Comparison of an unknown protein sample to polypeptides of knownmolecular weight allows a determination of the apparent molecular weightof the unknown protein sample (T. D. Brock and M. T. Madigan, Biology ofMicroorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Protein molecularweight standards are commercially available to assist in the estimationof molecular weights of unknown protein samples (New England BiolabsInc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758).However, the molecular weight standards may not correspond closelyenough in size to the unknown sample protein to allow an accurateestimation of apparent molecular weight.

The difficulty in estimation of molecular weight is compounded in thecase of proteins that are subjected to fragmentation by chemical orenzymatic means (A. L. Lehninger, Biochemistry 106-108 (Worth Books, 2ded. 1981)). Chemical fragmentation can be achieved by incubation of aprotein with a chemical, such as cyanogen bromide, which leads tocleavage of the peptide bond on the carboxyl side of methionine residues(E. Gross, Methods in Enz. 11:238-255, 1967). Enzymatic fragmentation ofa protein can be achieved by incubation of a protein with a proteasethat cleaves at multiple amino acid residues (D. W. Cleveland et al., J.Biol. Chem. 252:1102-1106, 1977). Enzymatic fragmentation of a proteincan also be achieved by incubation of a protein with a protease, such asAchromobacter protease I (F. Sakiyama and A. Nakata, U.S. Pat. No.5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50, 1981; T.Masaki et al., Biochim. Biophys. Acta 660:51-55, 1981), which leads tocleavage of the peptide bond on the carboxyl side of lysine residues.The molecular weights of the fragmented peptides can cover a large rangeof molecular weights and the peptides can be numerous. Variations in thedegree of fragmentation can also be accomplished (D. W. Cleveland etal., J. Biol. Chem. 252:1102-1106, 1977).

The unique nature of the composition of a protein with regard to itsspecific amino acid constituents results in a unique positioning ofcleavage sites within the protein. Specific fragmentation of a proteinby chemical or enzymatic cleavage results in a unique “peptidefingerprint” (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106,1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently,cleavage at specific sites results in reproducible fragmentation of agiven protein into peptides of precise molecular weights. Furthermore,these peptides possess unique charge characteristics that determine theisoelectric pH of the peptide. These unique characteristics can beexploited using a variety of electrophoretic and other techniques (T. D.Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall,6d ed. 1991)).

When a peptide fingerprint of an unknown protein is obtained, this canbe compared to a database of known proteins to assist in theidentification of the unknown protein (W. J. Henzel et al., Proc. Natl.Acad. Sci. USA 90:5011-5015, 1993; B. Thiede et al., Electrophoresis1996, 17:588-599, 1996). A variety of computer software programs areaccessible via the Internet to the skilled artisan for the facilitationof such comparisons, such as MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.emblheiedelberg.de...deSearch/FR_PeptideSearchForm.html), andProFound (Internetsite:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). Theseprograms allow the user to specify the cleavage agent and the molecularweights of the fragmented peptides within a designated tolerance. Theprograms compare these molecular weights to protein databases to assistin the elucidation of the identity of the sample protein. Accurateinformation concerning the number of fragmented peptides and the precisemolecular weight of those peptides is required for accurateidentification. Therefore, increasing the accuracy in the determinationof the number of fragmented peptides and the precise molecular weight ofthose peptides should result in enhanced success in the identificationof unknown proteins.

Fragmentation of proteins is further employed for the production offragments for amino acid composition analysis and protein sequencing (P.Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskom et al.,Electrophoresis 1988, 9:830-838, 1988), particularly the production offragments from proteins with a “blocked” N-terminus. In addition,fragmentation of proteins can be used in the preparation of peptides formass spectrometry (W. J. Henzel et al., Proc. Natl. Acad Sci. USA90:5011-5015, 1993; B. Thiede et al., Electrophoresis 1996, 17:588-599,1996), for immunization, for affinity selection (R. A. Brown, U.S. Pat.No.5,151,412), for determination of modification sites (e.g.phosphorylation), for generation of active biological compounds (T. D.Brock and M. T. Madigan, Biology ofMicroorganisms 300-301 (PrenticeHall, 6d ed. 1991)), and for differentiation of homologous proteins (M.Brown et al., J. Gen. Virol. 50:309-316, 1980).

In view of the continuing interest in protein research and theelucidation of protein structure and properties, there exists a need inthe art for polypeptides having kinase function or suitable for use inpeptide fragmentation studies and in molecular weight measurements.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these needs in the art. The inventionencompasses an isolated human nucleic acid molecule comprising the DNAsequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15 and an isolated humannucleic acid molecule encoding the amino acid sequence of SEQ ID NO:7,8, 9, 10, 11, 12, 14, or 16. The invention also encompasses nucleic acidmolecules complementary to these sequences. As such, the inventionincludes double-stranded nucleic acid molecules comprising the DNAsequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15 and isolated nucleicacid molecules encoding the amino acid sequence of SEQ ID NO:7, 8, 9,10, 11, 12, 14 or 16. Both single-stranded and double-stranded RNA andDNA nucleic acid molecules are encompassed by the invention. Thesemolecules can be used to detect both single-stranded and double-strandedRNA and DNA variants encompassed by the invention. A double-stranded DNAprobe allows the detection of nucleic acid molecules equivalent toeither strand of the nucleic acid molecule. Isolated nucleic acidmolecules that hybridize to a denatured, double-stranded DNA comprisingthe DNA sequence of SEQ ID NO:1, 2,3, 4, 5,6, 13, or 15, or an isolatednucleic acid molecule encoding the amino acid sequence of SEQ ID NO:7,8, 9, 10, 11, 12, 14, or 16 are within the invention. A preferred set ofhybridization conditions are those of moderate stringency: in 50%formamide and 6×SSC, at 42° C. with washing conditions of 60° C.,0.5×SSC, 0.1% SDS.

The invention further encompasses isolated nucleic acid moleculesderived by in vitro mutagenesis from SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or15. In vitro mutagenesis would include numerous techniques known in theart including, but not limited to, site-directed mutagenesis, randommutagenesis, and in vitro nucleic acid synthesis. The invention alsoencompasses isolated nucleic acid molecules degenerate from SEQ ID NO:1,2, 3, 4, 5, or 6 (and the resulting amino acid sequence) as a result ofthe genetic code, isolated nucleic acid molecules that are allelicvariants of human DNA of the invention, or a species homolog of DNA ofthe invention. The invention also encompasses recombinant vectors thatdirect the expression of these nucleic acid molecules and host cellstransformed or transfected with these vectors. In addition, theinvention encompasses methods of using the nucleic acid noted above inassays to identify chromosomes, map human genes, and study tumors.

The invention also encompasses isolated polypeptides encoded by thesenucleic acid molecules, including isolated polypeptides having amolecular weights as determined by SDS-PAGE, isolated polypeptides innon-glycosylated form, and fragments thereof. The invention furtherincludes synthetic polypeptides encoded by these nucleic acid molecules.Peptides and fragments of these polypeptides, however derived, are alsopart of the invention and may be produced by any standard means, fromchemical, enzymatic, recombinant, or synthetic methods. Isolatedpolyclonal or monoclonal antibodies that bind to these polypeptides areencompassed by the invention. The invention further encompasses methodsfor the production of polypeptides having kinase functions includingculturing a host cell under conditions promoting expression andrecovering the polypeptide from the culture medium. Especially, theexpression of polypeptides having kinase functions in bacteria, yeast,plant, insect, and animal cells is encompassed by the invention.

In general, the polypeptides of the invention having kinase function canbe used to phosphorylate target proteins and to radiolabeled targetproteins with ³²P. In addition, the polypeptides of the invention havingkinase function can be used to identify proteins having a phosphateactivity.

In addition, assays utilizing polypeptides having kinase functions toscreen for potential inhibitors of activity associated with polypeptidecounter-structure molecules, and methods of using polypeptides havingkinase functions as therapeutic agents for the treatment of diseasesmediated by polypeptide counter-structure molecules are encompassed bythe invention. Methods of using polypeptides having kinase functions inthe design of inhibitors thereof are also an aspect of the invention.The invention further encompasses use of polypeptides of the inventionto screen for agonists and antagonists.

The invention further encompasses the fragmented peptides produced frompolypeptides of the invention by chemical or enzymatic treatment. Inaddition, the polypeptides of the invention and fragmented peptidesthereof, wherein at least one of the sites necessary for fragmentationby chemical or enzymatic means has been mutated, are an aspect of theinvention.

The invention further includes a method for using these polypeptides andfragmented peptides thereof as molecular weight markers that allow theestimation of the molecular weight of a protein or a fragmented proteinsample. The invention also encompasses a method for the visualization ofthe molecular weight markers of the invention thereof usingelectrophoresis. The invention further encompasses methods for using thepolypeptides of the invention and fragmented peptides thereof asmarkers, which aid in the determination of the isoelectric point of asample protein. The invention also encompasses methods for usingpolypeptides of the invention and fragmented peptides thereof ascontrols for establishing the extent of fragmentation of a proteinsample.

Further encompassed by this invention are kits to aid the determinationof molecular weights of a sample protein utilizing polypeptide molecularweight markers of the invention, fragmented peptides thereof, and formsof these polypeptide molecular weight markers, wherein at least one ofthe sites necessary for fragmentation by chemical or enzymatic means hasbeen mutated.

DETAILED DESCRIPTION OF THE INVENTION

The protein kinases are a large family of enzymes, many of which mediatethe response of eukaryotic cells to external stimuli. In recent years,members of the protein kinase family have been discovered at anaccelerated pace. The surge in the number of known protein kinases hasbeen largely due to the advent of gene cloning and sequencingtechniques. Amino acid sequences deduced from nucleotide sequences areconsidered to represent protein kinases if they include certain keyresidues that are highly conserved in the protein kinase catalyticdomain. A cDNA encoding a human polypeptide has been isolated and is setforth in SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15. This discovery of thecDNA encoding human polypeptides having kinase functions enablesconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides having kinase functions; host cells transfected ortransformed with the expression vectors; biologically active humanpolypeptides having kinase functions, and molecular weight markers asisolated and purified proteins; and antibodies immunoreactive withpolypeptides of the invention.

More particularly, the invention relates to certain nucleotidesequences. A “nucleotide sequence” refers to a polynucleotide moleculein the form of a separate fragment or as a component of a larger nucleicacid construct, that has been derived from DNA or RNA isolated at leastonce in substantially pure form (i.e., free of contaminating endogenousmaterials) and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

Particularly preferred nucleotide sequences of the invention include thefollowing:

NAME: HH0900-BF04 DNA Nucleotide sequence:GTACGCCATGAAGGTGCTGCGCAAGGCGGCGCTGGTGCAGCGCGCCAAGA (SEQ ID NO:1)CGCAAGAGCACACGCGCACCGAGCGCTCGGTGCTGGAGCTGGTGCGCCAGGCGCCCTTCCTGGTCACGCTGCACTACGCTTTCCAGACGGATGCCAAGCTGCACCTCATCCTGGACTATGTGAGCGGCGGG; NAME: HH2046-BF04 DNA Nucleotidesequence: CCCGAGAGGTGCCACATCAGACCGCCTCCGACTTCGTGCGGGACTCGGCG (SEQ IDNO:2) GCCAGCCACCAGGCGGAGCCCGAGGCGTACGAGCGGCGCGTGTGCTTCCTGCTTCTGCAACTCTGCAACGGGCTGGAGCACCTGAAGGAGCACGGGATCATCCACCGGGACCTGTGCCTGGAGAACCTGCTGCTGGTGCACTGCACCCTCCAGGCCGGCCCCGGGCCCGCC; Name: JJ503-KS DNA nucleotide sequenceCGGGCAGGGCTGGAGCTGGGCTGGGATCCCGAGCTCGGCAGCAGCGCAGCGGGCCGGCCCACCTGCTGGTGC(SEQ ID NO:3)CCTGGAGGCTCTGAGCCCCGGCGGCGCCCGGGCCCACGCGGAACGACGGGGCGAGATGCGAGCCACCCCTCTGGCTGCTCCTGCGGGTTCCCTGTCCAGGAAGAAGCGGTTGGAGTTGGATGACAACTTAGATACCGAGCGTCCCGTCCAGAAACGAGCTCGAAGTGGGCCCCAGCCCAGACTGCCCCCCTGCCTGTTGCCCCTGAGCCCACCTACTGCTCCAGATCGTGCAACTGCTGTGGCCACTGCCTCCCGTCTTGGGCCCTATGTCCTCCTGGAGCCCGAGGAGGGCGGGCGGGCCTACCAGGCCCTGCACTGCCCTACAGGCACTGAGTATACCTGCAAGGTGTACCCCGTCCAGGAAGCCCTGGCCGTGCTGGAGCCCTACGCGCGGCTGCCCCCGCACAAGCATGTGGCTCGGCCCACTGAGGTCCTGGCTGGTACCCAGCTCCTCTACGCCTTTTTCACTCGGACCCATGGGGACATGCACAGCCTGGTGCGAAGCCGCCACCGTATCCCTGAGCCTGAGGCTGCCGTGCTCTTCCGCCAGATGGCCACCGCCCTGGCGCACTGTCACCAGCACGGTCTGGTCCTGCGTGATCTCAAGCTGTGTCGCTTTGTCTTCGCTGACCGTGAGAGGAAGAAGCTGGTGCTGGAGAACCTGGAGGACTCCTGCGTGCTGACTGGGCCAGATGATTCCCTGTGGGACAAGCACGCGTGCCCAGCCTACGTGGGACCTGAGATACTCAGCTCACGGGCCTCATACTCGGGCAAGGCAGCCGATGTCTGGAGCCTGGGCGTGGCGCTCTTCACCATGCTGGCCGGCCACTACCCCTTCCAGGACTCGGAGCCTGTCCTGCTCTTCGGCAAGATCCGCCGCGGGGCCTACGCCTTGCCTGCAGGCCTCTCGGCCCCTGCCCGCTGTCTGGTTCGCTGCCTCCTTCGTCGGGAGCCAGCTGAACGGCTCACAGCCACAGGCATCCTCCTGCACCCCTGGCTGCGACAGGACCCGA; Name: QQ1249-BF04 DNA Nucleotide sequence:CAGCGAGAAGCCGACATGCATCGCCTCTTCAATCACCCCAACATCCTTCG (SEQ ID NO:4)CCTCGTGGCTTACTGTCTGAGGGAACGGGGTGCTAAGCATGAGGCCTGGCTGCTGCTACCATTCTTCAAGAGAGGTACGCTGTGGAATGAGATAGAAAGGCTGAAGGACAAAGGCAACTTCCTGACCGAGGATCAAATCCTTTGGCTGCTGCTGGGGATCTGCAGAGGCCTTGAGGCCATTCATGCCAAGGGTTATGCCTACAGAGACTTGAAGCCCACCAATATATTGCTTGGAGATGAGGGGCAGCCAGTTTTAATGGACTTGGGTTCCATGAATCAAGCATGCATCCATGTGGAGGGCTCCCGCCAGGCTCTGACCCTGCAGGACTGGGCAGCCC; Name: QQ3351-BF04 DNA Nucleotidesequence:ATGCTAACTAGTTTAAACAGATCTTGGAACGAGACGACCTGCTGTGGAAGAGCGAGCTTTTTGGAACTGTGC(SEQ ID NO:5)ACGGGACAGATTGGACGCACACCCCTCGGGAGGCGCGAAGGCATGGAAAATTTGAAGCATATTATCACCCTTGGCCAGGTCATCCACAAACGGTGTGAAGAGATGAAATACTGCAAGAAACAGTGCCGGCGCCTGGGCCACCGCGTCCTCGGCCTGATCAAGCCTCTGGAGATGCTCCAGGACCAAGGAAAGAGGAGCGTGCCCTCTGAGAAGTTAACCACAGCCATGAACCGCTTCAAGGCTGCCCTGGAGGAGGCTAATGGGGAGATAGAAAAGTTCAGCAATAGATCCAATATCTGCAGGTTTCTAACAGCAAGCCAGGACAAAATACTCTTCAAGGACGTGAACAGGAAGCTGAGTGATGTCTGGAAGGAGCTCTCGCTGTTACTTCAGGTTGAGCAACGCATGCCTGTTTCACCCATAAGCCAAGGAGCGTCCTGGGCACAGGAAGATCAGCAGGATGCAGACGAAGACAGGCGAGCTTTCCAGATGCTAAGAAGAGATAATGAAAAAATAGAAGCTTCACTGAGACGATTAGAAATCAACATGAAAGAAATCAAGGAAACTTTGAGGCAGTATTTACCACCAAAATGCATGCAGGAGATCCCGCAAGAGCAAATCAAGGAGATCAAGAAGGAGCAGCTTTCAGGATCCCCGTGGATTCTGCTAAGGGAAAATGAAGTCAGCACACTTTATAAAGGAGAATACCACAGAGCTCCAGTGGCCATAAAAGTATTCAAAAAACTCCAGGCTGGCAGCATTGCAATAGTGAGGCAGACTTTCAATAAGGAGATCAAAACCATGAAGAAATTCGAATCTCCCAACATCCTGCGTATATTTGGGATTTGCATTGATGAAACAGTGACTCCGCCTCAATTCTCCATTGTCATGGAGTACTGTGAACTCGGGACCCTGAGGGAGCTGTTGGATAGGGAAAAAGACCTCACACTTGGCAAGCGCATGGTCCTAGTCCTGGGGGCAGCCCGAGGCCTATACCGGCTACACCATTCAGAAGCACCTGAACTCCACGGAAAAATCAGAAGCTCAAACTTCCTGGTAACTCAAGGCTACCAAGTGAAGCTTGCAGGATTTGAGTTGAGGAAAACACAGACTTCCATGAGTTTGGGAACTACGAGAGAAAAGACAGACAGAGTCAAATCTACAGCATATCTCTCACCTCAGGAACTGGAAGATGTATTTTATCAATATGATGTAAAGTCTGAAATATACAGCTTTGGAATCGTCCTCTGGGAAATCGCCACTGGAGATATCCCGTTTCAAGGCTGTAATTCTGAGAAGATCCGCAAGCTGGTGGCTGTGAAGCGGCAGCAGGAGCCACTGGGTGAAGACTGCCCTTCAGAGCTGCGGGAGATCATTGATGAGTGCCGGGCAGCAGGTCGTCTCGTTCCAAGATCTGTAGCGGCCGCCCGGGCCGTCGACGTTTAAACGCGTGGCCCTCGAGAGGTTTTCCGATCCGGTCGAT, and Name: SS1771 Nucleotidesequence: CTTCCCGCTG GACGTGGAGT ACGGAGGCCC AGACCGGAGG TGCCCGCCTC (SEQ IDNO:6) CGCCCTACCC GAAGCACCTG CTGCTGCGCA GCAAGTCGGA GCAGTACGAC CTGGACAGCCTGTGCGCAGG CATGGAGCAG AGCCTCCGTG CGGGCCCCAA CGAGCCCGAG GGCGGCGACAAGAGCCGCAA AAGCGCCAAG GGGGACAAAG GCGGAAAGGA TAAAAAGCAG ATTCAGACCTCTCCCGTTCC CGTCCGCAAA AACAGCAGAG ACGAAGAGAA GAGAGAGTCA CGCATCAAGAGCTACTCGCC ATACGCCTTT AAGTTCTTCA TGGAGCAGCA CGTGGAGAAT GTCATCAAAACCTACCAGCA GAAGGTTAAC CGGAGGCTGC AGCTGGAGCA AGAAATGGCC AAAGCTGGACTCTGTGAAGC TGAGCAGGAG CAGATGCGGA AGATCCTCTA CCAGAAAGAG TCTAATTACAACAGGTTAAA GAGGGCCAAG ATGGACAAGT CTATGTTTGT CAAGATCAAA ACCCTGGGGATCGGTGCCTT TGGAGAAGTG TGCCTTGCTT GTAAGGTGGA CACTCACGCC CTGTACGCCATGAAGACCCT AAGGAAAAAG GATGTCCTGA ACCGGAATCA GGTGGCCCAC GTCAAGGCCGAGAGGGACAT CCTGGCCGAG GCAGACAATG AGTGGGTGGT CAAACTCTAC TACTCCTTCCAAGACAAAGA CAGCCTGTAC TTTGTGATGG ACTACATCCC TGGTGGGGAC ATGATGAGCCTGCTGATCCG GATGGAGGTC TTCCCTGAGC ACCTGGCCCG GTTCTACATC GCAGAGCTGACTTTGGCCAT TGAGAGTGTC CACAAGATGG GCTTCATCCA CCGAGACATC AAGCCTGATAACATTTTGAT AGATCTGGAT GGTCACATTA AACTCACAGA TTTCGGCCTC TGCACTGGGTTCAGGTGGAC TCACAATTCC AAATATTACC AGAAAGGGAG CCATGTCAGA CAGGACAGCATGGAGCCCAG CGACCTCTGG GATGATGTGT CTAACTGTCG GTGTGGGGAC AGGCTGAAGACCCTAGAGCA GAGGGCGCGG AAGCAGCACC AGAGGTGCCT GGCACATTCA CTGGTGGGGACTCCAAACTA CATCGCACCC GAGGTGCTCC TCCGCAAAGG GTACACTCAA CTCTGTGACTGGTGGAGTGT TGGAGTGATT CTCTTCGAGA TGCTGGTGGG GCAGCCGCCC TTTTTGGCACCTACTCCCAC AGAAACCCAG CTGAAGGTGA TCAACTGGGA GAACACGCTC CACATTCCAGCCCAGGTGAA GCTGAGCCCT GAGGCCAGGG ACCTCATCAC CAAGCTGTGC TGCTCCGCAGACCACCGCCT GGGGCGGAAT GGGGCCGATG ACCTGAAGGC CCACCCCTTC TTCAGCGCCATTGACTTCTC CAGTGACATC CGGAAGCATC CAGCCCCCTA CGTTCCCACC ATCAGCCACCCCATGGAG Name: S51771k Nucleotide sequence:TCCCGCTGGACGTGGAGTACGGAGGCCCAGACCGGAGGTGCCCGCCTCCGCCCTACCCGAAGCACCTGCTGC(SEQ ID NO:15)TGCGCAGCAAGTCGGAGCAGTACGACCTGGACAGCCTGTGCGCAGGCATGGAGCAGAGCCTCCGTGCGGGCCCCAACGAGCCCGAGGGCGGCGACAAGAGCCGCAAAAGCGCCAAGGGGGACAAAGGCGGAAAGGATAAAAAGCAGATTCAGACCTCTCCCGTTCCCGTCCGCAAAAACAGCAGAGACGAAGAGAAGAGAGAGTCACGCATCAAGAGCTACTCGCCATACGCCTTTAAGTTCTTCATGGAGCAGCACGTGGAGAATGTCATCAAAACCTACCAGCAGAAGGTTAACCGGAGGCTGCAGCTGGAGCAAGAAATGGCCAAAGCTGGACTCTGTGAAGCTGAGCAGGAGCAGATGCGGAAGATCCTCTACCAGAAAGAGTCTAATTACAACAGGTTAAAGAGGGCCAAGATGGACAAGTCTATGTTTGTCAAGATCAAAACCCTGGGGATCGGTGCCTTTGGAGAAGTGTGCCTTGCTTGTAAGGTGGACACTCACGCCCTGTACGCCATGAAGACCCTAAGGAAAAAGGATGTCCTGAACCGGAATCAGGTGGCCCACGTCAAGGCCGAGAGGGACATCCTGGCCGAGGCAGACAATGAGTGGGTGGTCAACCTCTACTACTCCTTCCAAGACAAAGACAGCCTGTACTTTGTGATGGACTACATCCCTGGTGGGGACATGATGAGCCTGCTGATCCGGATGGAGGTCTTCCCTGAGCACCTGGCCCGGTTCTACATCGCAGAGCTGACTTTGGCCATTGAGAGTGTCCACAAGATGGGCTTCATCCACCGAGACATCAAGCCTGATAACATTTTGATAGATCTGGATGGTCACATTAAACTCACAGATTTCGGCCTCTGCACTGGGTTCAGGTGGACTCACAATTCCAAATATTACCAGAAAGGGAGCCATGTCAGACAGGACAGCATGGAGCCCAGCGACCTCTGGGATGATGTGTCTAACTGTCGGTGTGGGGACAGGCTGAAGACCCTAGAGCAGAGGGCGCGGAAGCAGCACCAGAGGTGCCTGGCACATTCACTGGTGGGGACTCCAAACTACATCGCACCCGAGGTGCTCCTCCGCAAAGGGTACACTCAACTCTGTGACTGGTGGAGTGTTGGAGTGATTCTCTTCGAGATGCTGGTGGGGCAGCCGCCCTTTTTGGCACCTACTCCCACAGAAACCCAGCTGAAGGTGATCAACTGGGAGAACACGCTCCACATTCCAGCCCAGGTGAAGCTGAGCCCTGAGGCCAGGGACCTCATCACCAAGCTGTGCTGCTCCGCAGACCACCGCCTGGGGCGGAATGGGGCCGATGACCTGAAGGCCCACCCCTTCTTCAGCGCCATTGACTTCTCCAGTGACATCCGGAAGCATCCAGCCCCCTACGTTCCCACCATCAGCCACCCCATGGACACCTCGAATTTCGACCCCGTAGATGAAGAAAGCCCTTGGAACGATGCCAGCGAAGGTAGCACCAAGGCCTGGGACACACTCACCTCGCCCAATAACAAGCATCCTGAGCACGCATTTTACGAATTCACCTTCCGAAGGTTCTTTGATGACAATGGCTACCCCTTTCGATGCCCAAAGCCTTCAGGAGCAGAAGCTTCACAGGCTGAGAGCTCAGATTTAGAAAGCTCTGATCTGGTGGATCAGACTGAAGGCTGCCAGCCTGTGTACGTGTAGATGGGGGCCAGGCACCCCCACCACTCGCTGCCTCCCAGGTCAGGGTCCCGGAGCCGGTGCCCTCACAGGCCAATAGGGAAGCCGAGGGCTGTTTTGTTTTAAATTAGTCCGTCGATTACTTCACTTGAAATTCTGCTCTTCACCAAGAAAACCCAAACAGGACACTTTTGAAAACAGCGGTGCCGCGAATTC.

With the continued increase in the number of known eukaryotic proteinkinases, a suitable classification scheme is one based on comparingcatalytic-domain sequences. It follows that protein kinases with similarcatalytic domains will tend also to have similar enzymatic andregulatory properties. The nucleotide sequences of the invention encode,respectively, the following polypeptides having kinase function:

NAME: HH0900-BF04 Polypeptide

Translation in relevant reading frame (3′-5′ Frame 3):

YAMKVLRKAALVQRAKTQEHTRTERSVLELVRQAPFLVTLHYAFQTDAKLHLILDY VSGG (SEQ IDNO:7);

The above sequence is consistent with the consensus sequence ofsubdomains II through V of the eukaryotic protein kinase superfamily.This sequence is the sequence of a polypeptide having kinase activityencoded by the nucleotide sequence of SEQ ID NO:1.

NAME: HH2040-BF04 Polypeptide

Translation in relevant reading frame (5′-3′ Frame 2):

REVPHQTASDFVRDSAASHQAEPEAYERRVCFLLLQLCNGLEHLKEHGIIHRDLCLENLLLVHCTLQAGPGPA (SEQ ID NO:8);

The above sequence is consistent with the consensus sequence ofsubdomains V, VIA and VIB of the eukaryotic protein kinase superfamily.This sequence is the sequence of a polypeptide having kinase activityencoded by the nucleotide sequence of SEQ ID NO:2.

Name: JJ503-KS Polypeptide

Translation in relevant reading frame (5′-3′ frame 1):

(SEQ ID NO:9) GQGWSWAGIPSSAAAQRAGPPAGALEALSPGGARAHAERRGEMRATPLAAPAGSLSRKKRLELDDNLDTERPVQKRARSGPQPRLPPCLLPLSPPTAPDRATAVATASRLGPYVLLEPEEGGRAYQALHCPTGTEYTCKVYPVQEALAVLEPYARLPPHKHVARPTEVLAGTQLLYAFFTRTHGDMHSLVRSRHRIPEPEAAVLFRQMATALAHCHQHGLVLRDLKLCRFVFADRERKKLVLENLEDSCVLTGPDDSLWDKHACPAYVGPEILSSRASYSGKAADVWSLGVALFTMLAGHYPFQDSEPVLLFGKIRRGAYALPAGLSAPARCLVRCLLRREPAERLTATG ILLHPWLRQD;

The sequence is consistent with kinase domains VIA through XI, thoughthe homology is imperfect. This sequence is the sequence of apolypeptide having kinase activity encoded by the nucleotide sequence ofSEQ ID NO:3.

Name: QQ1249-BF04 Polypeptide

Translation in relevant reading frame (5′-3′ frame 3)

(SEQ ID NO:10) QREADMHRLFNHPNILRLVAYCLRERGAKHEAWLLLPFFKRGTLWNEIERLKDKGNFLTEDQILWLLLGICRGLEAIHAKGYAYRDLKPTNILLGDEGQPVLMDLGSMNQACIHVEGSRQALTLQDWAAQRCTISYRAPXLFSVQS

The above sequence is consistent with the consensus sequence ofsubdomains III-VIII of the eukaryotic protein kinase superfamily. Thissequence is the sequence of a polypeptide having kinase activity encodedby the nucleotide sequence of SEQ ID NO:4.

Name: QQ3351-BF04 Polypeptide

Translation in relevant frame (5′-3′ frame 1)

MLTSLNRSWNETTCCGRASFLELCTGQIGRTPLGRREGMENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHR(SEQ ID NO:11)VLGLIKPLEMLQDQGKRSVPSEKLTTAMNRFKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQGASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQIKEIKKEQLSGSPWILLREMEVSTLYKGEYHRAPVAIKVFKKLQAGSIAIVRQTFNKEIKTMKKFESPNILRIFGICIDETVTPPQFSIVMEYCELGTLRELLDREKDLTLGKRMVLVLGAARGLYRLHHSEAPELHGKIRSSNFLVTQGYQVKLAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAAGRLVPRSVAAARAVDV

The above sequence is believed to be full-length. However, the initialmethionine was not present in the clone sequenced but subsequently wasadded by PCR. Therefore, the natural sequence may comprise Leu-2 throughthe Val-505. This sequence is the sequence of a polypeptide havingkinase activity encoded by the nucleotide sequence of SEQ ID NO:5.

Name: SS1771

Translation in relevant frame (3′-5′ frame 3)

(SEQ ID NO:12) FPLDVEYGGPDRRCPPPPYPKHLLLRSKSEQYDLDSLCAGMEQSLRAGPNEPEGGDKSRKSAKGDKGGKDKKQIQTSPVPVRKNSRDEEKRESRIKSYSPYAFKFFMEQHVENVIKTYQQKVNRRLQLEQEMAKAGLCEAEQEQMRKILYQKESNYNRLKRAKMDKSMFVKIKTLGIGAFGEVCLACKVDTHALYAMKTLRKKDVLNRNQVAHVKAERDILAEADNEWVVKLYYSFQDKDSLYFVMDYIPGGDMMSLLIRMEVFPEHLARFYIAELTLAIESVHKMGFIHRDIKPDNILIDLDGHIKLTDFGLCTGFRWTHNSKYYQKGSHVRQDSMEPSDLWDDVSNCRCGDRLKTLEQRARKQHQRCLAHSLVGTPNYIAPEVLLRKGYTQLCDWWSVGVILFEMLVGQPPFLAPTPTETQLKVINWENTLHIPAQVKLSPEARDLITKLCCSADHRLGRNGADDLKAHPFFSAIDFSSDIRKHPAPYVPTISHPME

This sequence is the sequence of a polypeptide having kinase activityencoded by the nucleotide sequence of SEQ ID NO:6. The sequence may alsocomprise Pro-2 through Glu499.

Name: SS1771A

Translation in relevant frame (5′-3′ frame 3)

PLDVEYGGPDRRCPPPPYPKHLLLRSKSEQYDLDSLCAGMEQSLRAGPNEPEGGDKSRKSAKGDKGGKDKKQ(SEQ ID NO:16)IQTSPVPVRKNSRDEEKRESRIKSYSPYAFKFFMEQHVENVIKTYQQKVNRRLQLEQEMAKAGLCEAEQEQMRKILYQKESNYNRLKRAKMDKSMFVKIKTLGIGAFGEVCLACKVDTHALYAMKTLRKKDVLNRNQVAHVKAERDILAEADNEWVVKLYYSFQDKDSLYFVMDYIPGGDMMSLLIRMEVFPEHLARFYIAELTLAIESVHKMGFIHRDIKPDNILIDLDGHIKLTDFGLCTGFRWTHNSKYYQKGSHVRQDSMEPSDLWDDVSNCRCGDRLKTLEQRARKQHQRCLAHSLVGTPNYIAPEVLLRKGYTQLCDWWSVGVILFEMLVGQPPFLAPTPTETQLKVINWENTLHIPAQVKLSPEARDLITKLCCSADHRLGRNGADDLKAHPFFSAIDFSSDIRKHPAPYVPTISHPMDTSNFDPVDEESPWNDASEGSTKAWDTLTSPNNKHPEHAFYEFTFRRFFDDNGYPFRCPKPSGAEASQAESSDLESSDLVDQTEGCQPVYV

This sequence is the sequence of a polypeptide having kinase activityencoded by the nucleotide sequence of SEQ ID NO:1.

The invention also includes truncated forms of the nucleic acids andpolypeptides of the invention. In a preferred embodiment, the inventionincludes a truncated form of QQ3351-BF04, as follows:

Nucleotide sequence:

CTTGCAGGATTTGAGTTGAGGAAAACACAGACTTCCATGAGTTTGGGAACTACGAGAGAAAAGACAGACAGA(SEQ ID NO:13)GTCAAATCTACAGCATATCTCTCACCTCAGGAACTGGAAGATGTATTTTATCAATATGATGTAAAGTCTGAAATATACAGCTTTGGAATCGTCCTCTGGGAAATCGCCACTGGAGATATCCCGTTTCAAGGCTGTAATTCTGAGAAGATCCGCAAGCTGGTGGCTGTGAAGCGGCAGCAGGAGCCACTGGGTGAAGACTGCCCTTCAGAGCTGCGGGAGATCATTGATGAGTGCCGGGCCCATGATCCCTCTGTGCGGCCCTCTGTGGATGAAATCTTAAAGAAACTCTCCACCTTTTCTAAG

Translation in relevant frame (5′3′ frame 1):

(SEQ ID NO:14) LAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAHDPSVRPSVDEILKKLSTFSK

This sequence is the sequence of a polypeptide having kinase activityencoded by the nucleotide sequence of SEQ ID NO:13.

The polypeptides of the invention are useful for characterizing cell andtissue expression, understanding their roles in development or hormonalresponse, and identifying regulatory molecules and physiologicallyrelevant protein substrates.

As used herein, the term “polypeptides of the invention” refers to agenus of polypeptides that further encompasses proteins having the aminoacid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 14, or 16 as well asthose proteins having a high degree of similarity (at least 90%homology) with such amino acid sequences and which proteins arebiologically active. In addition, polypeptides of the invention refersto the gene products of the nucleotides of SEQ ID NO:1, 2, 3, 4, 5, 6,13 or 15.

The isolated and purified polypeptides of the invention have molecularweights of approximately 6883 (HH0900-BF04); 8168 (HH2040-BF04); 39,284(JJ503-KS); 16,718 (QQ1249-BF04); 58,001 (QQ3351-BF04); 57,381 (SS1771),and 67,331 (SS1771A) Daltons in the absence of glycosylation. It isunderstood that the molecular weight of these polypeptides can be variedby fusing additional peptide sequences to both the amino and carboxylterminal ends of polypeptides of the invention. Fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention can be used to enhance expression of thesepolypeptides or aid in the purification of the protein.

It is understood that fusions of additional peptide sequences at theamino and carboxyl terminal ends of polypeptides of the invention willalter some, but usually not all, of the fragmented peptides of thepolypeptides generated by enzymatic or chemical treatment.

It is understood that mutations can be introduced into polypeptides ofthe invention using routine and known techniques of molecular biology.It is further understood that a mutation can be designed so as toeliminate a site of proteolytic cleavage by a specific enzyme or a siteof cleavage by a specific chemically induced fragmentation procedure. Itis also understood that the elimination of the site will alter thepeptide fingerprint of polypeptides of the invention upon fragmentationwith the specific enzyme or chemical procedure.

The term “isolated and purified” as used herein, means that thepolypeptides or fragments of the invention are essentially free ofassociation with other proteins or polypeptides, for example, as apurification product of recombinant host cell culture or as a purifiedproduct from a non-recombinant source. The term “substantially purified”as used herein, refers to a mixture that contains polypeptides orfragments of the invention and is essentially free of association withother proteins or polypeptides, but for the presence of known proteinsthat can be removed using a specific antibody, and which substantiallypurified polypeptides or fragments thereof can be used as molecularweight markers. The term “purified” refers to either the “isolated andpurified” form of polypeptides of the invention or the “substantiallypurified” form of polypeptides of the invention, as both are describedherein.

A polypeptide “variant” as referred to herein means a polypeptidesubstantially homologous to native polypeptides of the invention, butwhich has an amino acid sequence different from that of nativepolypeptides (human, murine or other mammalian species) of the inventionbecause of one or more deletions, insertions or substitutions. Thevariant amino acid sequence preferably is at least 80% identical to anative polypeptide amino acid sequence, most preferably at least 90%identical. The percent identity can be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Variants can comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events, proteolytic cleavage of thepolypeptides, or transcription/translation from different alleles.Variations attributable to proteolysis include, for example, differencesin the N- or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from thepolypeptides (generally from 1-5 terminal amino acids) of the invention.

The polypeptides of the invention can also exist as oligomers, such ascovalently linked or non-covalently linked dimers or trimers. Oligomerscan be linked by disulfide bonds formed between cysteine residues ondifferent polypeptides.

In one embodiment of the invention, a polypeptide dimer is created byfusing polypeptides of the invention to the Fc region of an antibody(e.g., IgG1) in a manner that does not interfere with the biologicalactivity of these polypeptides. The Fc region preferably is fused to theC-terminus of a soluble polypeptide of the invention, to form an Fcfusion or an Fc polypeptide. The terms “Fc fusion protein” or “Fcpolypeptides” as used herein includes native and mutein forms, as wellas truncated Fc polypeptides containing the hinge region that promotesdimerization. Exemplary methods of making Fc polypeptides set forthabove are disclosed in U.S. Pat. Nos. 5,426,048 and 5,783,672 both ofwhich are incorporated herein by reference.

In a preferred embodiment, extracellular domains from transmembranebound kinases are fused to Fc portions of antibodies to produce solubleFc polypeptides. These constructs can function as binding sites for theligand that naturally binds the kinase receptor and thereby inhibitbinding of the ligand to the natural receptor.

General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the polypeptide:Fcfusion protein of the invention is inserted into an appropriateexpression vector. Polypeptide:Fc fusion proteins are allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between Fc polypeptides, yielding divalent polypeptides ofthe invention. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form a polypeptide oligomerwith as many as four polypeptides extracellular regions. Alternatively,one can link two soluble polypeptide domains with a peptide linker.

In one embodiment of this invention, the polypeptides of the inventionare produced by recombinant expression. In one preferred embodiment, theexpression of recombinant polypeptides having kinase functions can beaccomplished utilizing fusion of sequences encoding polypeptides havingkinase functions to sequences encoding another polypeptide to aid in thepurification of polypeptides of the invention. An example of such afusion is a fusion of sequences encoding a polypeptide having kinasefunctions to sequences encoding the product of the malE gene of thepMAL-c2 vector of New England Biolabs, Inc. Such a fusion allows foraffinity purification of the fusion protein, as well as separation ofthe maltose binding protein portion of the fusion protein from thepolypeptide of the invention after purification.

The insertion of DNA encoding the polypeptide having kinase functionsinto the pMAL-c2 vector can be accomplished in a variety of ways usingknown molecular biology techniques. The preferred construction of theinsertion contains a termination codon adjoining the carboxyl terminalcodon of the polypeptide of the invention. In addition, the preferredconstruction of the insertion results in the fusion of the aminoterminus of the polypeptide of the invention directly to the carboxylterminus of the Factor Xa cleavage site in the pMAL-c2 vector. A DNAfragment can be generated by PCR using DNA of the invention as thetemplate DNA and two oligonucleotide primers. Use of the oligonucleotideprimers generates a blunt-ended fragment of DNA that can be isolated byconventional means. This PCR product can be ligated together withpMAL-p2 (digested with the restriction endonuclease Xmn I) usingconventional means. Positive clones can be identified by conventionalmeans. Induction of expression and purification of the fusion proteincan be performed as per the manufacturer's instructions. Thisconstruction facilitates a precise separation of the polypeptide of theinvention from the fused maltose binding protein utilizing a simpleprotease treatment as per the manufacturer's instructions. In thismanner, purified polypeptide having kinase functions can be obtained.Furthermore, such a constructed vector can be easily modified usingknown molecular biology techniques to generate additional fusionproteins. It is understood, of course, that many different vectors andtechniques can be used for the expression and purification ofpolypeptides of the invention and that this embodiment in no way limitsthe scope of the invention.

Polypeptides of the invention can be subjected to fragmentation intopeptides by chemical and enzymatic means. Chemical fragmentationincludes the use of cyanogen bromide to cleave under neutral or acidicconditions such that specific cleavage occurs at methionine residues (E.Gross, Methods in Enz. 11:238-255, 1967). This can further includeadditional steps, such as a carboxymethylation step to convert cysteineresidues to an unreactive species. Enzymatic fragmentation includes theuse of a protease such as Asparaginylendopeptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta660:51-55, 1981). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Staphlococcus aureus V8 protease can cleave specifically onthe carboxyl side of the aspartic and glutamic acid residues presentwithin polypeptides (D. W. Cleveland, J. Biol. Chem. 3:1102-1106, 1977).Endoproteinase Asp-N can cleave specifically on the amino side of theasparagine residues present within polypeptides. Endoproteinase Lys-Ccan cleave specifically on the carboxyl side of the lysine residuespresent within polypeptides of the invention. Other enzymatic andchemical treatments can likewise be used to specifically fragment thesepolypeptides into a unique set of specific peptide molecular weightmarkers.

The resultant fragmented peptides can be analyzed by methods includingsedimentation, electrophoresis, chromatograpy, and mass spectrometry.The fragmented peptides derived from the polypeptides of the inventioncan serve as molecular weight markers using such analysis techniques toassist in the determination of the molecular weight of a sample protein.Such a molecular weight determination assists in the identification ofthe sample protein. Fragmented peptide molecular weight markers of theinvention are preferably at least 10 amino acids in size. Morepreferably, these fragmented peptide molecular weight markers arebetween 10 and 100 amino acids in size. Even more preferable arefragmented peptide molecular weight markers between 10 and 50 aminoacids in size and especially between 10 and 35 amino acids in size. Mostpreferable are fragmented peptide molecular weight markers between 10and 20 amino acids in size.

Furthermore, analysis of the progressive fragmentation of thepolypeptides of the invention into specific peptides (D. W. Cleveland etal., J. Biol. Chem. 252:1102-1106, 1977), such as by altering the timeor temperature of the fragmentation reaction, can be used as a controlfor the extent of cleavage of a sample protein. For example, cleavage ofthe same amount of polypeptide and sample protein under identicalconditions can allow for a direct comparison of the extent offragmentation. Conditions that result in the complete fragmentation ofthe polypeptide can also result in complete fragmentation of the sampleprotein.

In addition, the polypeptides and fragmented peptides of the inventionpossess unique charge characteristics and, therefore, can serve asspecific markers to assist in the determination of the isoelectric pointof a sample protein or fragmented peptide using techniques such asisoelectric focusing. The technique of isoelectric focusing can befurther combined with other techniques such as gel electrophoresis tosimultaneously separate a protein on the basis of molecular weight andcharge. An example of such a combination is that of two-dimensionalelectrophoresis (T. D. Brock and M. T. Madigan, Biology ofMicroorganisms 76-77 (Prentice Hall, 6d ed. 1991)). These polypeptidesand fragmented peptides thereof can be used in such analyses as markersto assist in the determination of both the isoelectric point andmolecular weight of a sample protein or fragmented peptide.

Kits to aid in the determination of apparent molecular weight andisoelectric point of a sample protein can be assembled from thepolypeptides and peptide fragments of the invention. Kits also serve toassess the degree of fragmentation of a sample protein. The constituentsof such kits can be varied, but typically contain the polypeptide andfragmented peptide molecular weight markers. Also, such kits can containthe polypeptides wherein a site necessary for fragmentation has beenremoved. Furthermore, the kits can contain reagents for the specificcleavage of the polypeptide and the sample protein by chemical orenzymatic cleavage. Kits can further contain antibodies directed againstpolypeptides or fragments thereof of the invention.

The isolated and purified polypeptides of the invention have molecularweights of approximately 6883 (HH0900-BF04); 8168 (HH2040-BF04); 39,284(JJ503-KS); 16,718 (QQ1249-BF04); 58,001 (QQ3351-BF04); 57,381 (SS1771),and 67,331 (SS1771A) Daltons in the absence of glycosylation. Thepolypeptide of the invention, together with a sample protein, can beresolved by denaturing polyacrylamide gel electrophoresis byconventional means (U. K. Laemmli, Nature 227:680-685, 1970) in twoseparate lanes of a gel containing sodium dodecyl sulfate and aconcentration of acrylamide between 6-20%. Proteins on the gel can bevisualized using a conventional staining procedure. The polypeptidemolecular weight markers of the invention can be used as molecularweight marker in the estimation of the apparent molecular weight of thesample protein. The unique amino acid sequence of SEQ ID NO:7, 8, 9, 10,11, 12, and 16 correspond to molecular weight of approximately 6883;8168; 39,284; 16,718; 58,001; 57,381; or 67,331 Daltons, respectively.Therefore, the polypeptide molecular weight markers serve particularlywell as a molecular weight marker for the estimation of the apparentmolecular weight of sample proteins that have apparent molecular weightsclose to 6883; 8168; 39,284; 16,718; 58,001; 57,381; or 67,331 Daltons.The use of these polypeptide molecular weight markers allows increasedaccuracy in the determination of apparent molecular weight of proteinsthat have apparent molecular weights close to 6883; 8168; 39,284;16,718; 28,982; or 57,381 Daltons. It is understood of course that manydifferent techniques can be used for the determination of the molecularweight of a sample protein using polypeptides of the invention, and thatthis embodiment in no way limits the scope of the invention.

Another preferred embodiment of the invention is the use of polypeptidesand fragmented peptides of the invention as molecular weight markers toestimate the apparent molecular weight of a sample protein by gelelectrophoresis. These fragmented peptides can be generated methods wellknown in the art such as chemical fragmentation. Isolated and purifiedpolypeptides of the invention can be treated with cyanogen bromide underconventional conditions that result in fragmentation of the polypeptidemolecular weight marker by specific hydrolysis on the carboxyl side ofthe methionine residues within the polypeptides of the invention (E.Gross, Methods in Enz. 11:238-255, 1967). Due to the unique amino acidsequence of the polypeptides of the invention, the fragmentation ofpolypeptide molecular weight markers with cyanogen bromide generates aunique set of fragmented peptide molecular weight markers. Thedistribution of methionine residues determines the number of amino acidsin each peptide and the unique amino acid composition of each peptidedetermines its molecular weight. Polypeptide molecular weight markers ofthe invention can be analyzed by methods including sedimentation, gelelectrophoresis, chromatography, and mass spectrometry.

The fragmented peptide molecular weight markers of the invention,together with a sample protein, can be resolved by denaturingpolyacrylamide gel electrophoresis by conventional means in two separatelanes of a gel containing sodium dodecyl sulfate and a concentration ofacrylamide between 10-20%. Proteins on the gel can be visualized using aconventional staining procedure. The fragmented peptide molecular weightmarkers of the invention can be used as molecular weight markers in theestimation of the apparent molecular weight of the sample protein. Theunique amino acid sequence of each marker specifies a molecular weight.Therefore, the fragmented peptide molecular weight markers serveparticularly well as molecular weight markers for the estimation of theapparent molecular weight of sample proteins that have similar apparentmolecular weights. Consequently, the use of these fragmented peptidemolecular weight markers allows increased accuracy in the determinationof apparent molecular weight of proteins.

Polypeptides on the membrane can be visualized using two differentmethods that allow a discrimination between the sample protein and themolecular weight markers. Polypeptide or fragmented peptide molecularweight markers of the invention can be visualized using antibodiesgenerated against these markers and conventional immunoblottingtechniques. This detection is performed under conventional conditionsthat do not result in the detection of the sample protein. It isunderstood that it may not be possible to generate antibodies againstall polypeptide fragments of the invention, since small peptides may notcontain immunogenic epitopes. It is further understood that not allantibodies will work in this assay; however, those antibodies which areable to bind polypeptides and fragments of the invention can be readilydetermined using conventional techniques.

The sample protein is visualized using a conventional stainingprocedure. The molar excess of sample protein to polypeptide orfragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects thesample protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of sample protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of sample protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

The polypeptide or fragmented peptide molecular weight markers of theinvention can be used as molecular weight and isoelectric point markersin the estimation of the apparent molecular weight and isoelectric pointof the sample protein. These polypeptide or fragmented peptide molecularweight markers serve particularly well as molecular weight andisoelectric point markers for the estimation of apparent molecularweights and isoelectric points of sample proteins that have apparentmolecular weights and isoelectric points close to that of thepolypeptide or fragmented peptide molecular weight markers of theinvention. The ability to simultaneously resolve these polypeptide orfragmented peptide molecular weight markers and the sample protein underidentical conditions allows for increased accuracy in the determinationof the apparent molecular weight and isoelectric point of the sampleprotein. This is of particular interest in techniques, such as twodimensional electrophoresis, where the nature of the procedure dictatesthat any markers should be resolved simultaneously with the sampleprotein.

In another embodiment, polypeptide or fragmented peptide molecularweight markers of the invention can be used as molecular weight andisoelectric point markers in the estimation of the apparent molecularweight and isoelectric point of fragmented peptides derived by treatmentof a sample protein with a cleavage agent. It is understood of coursethat many techniques can be used for the determination of molecularweight and isoelectric point of a sample protein and fragmented peptidesthereof using these polypeptide molecular weight markers and peptidefragments thereof and that this embodiment in no way limits the scope ofthe invention.

Polypeptide molecular weight markers encompassed by invention can havevariable molecular weights, depending upon the host cell in which theyare expressed. Glycosylation of polypeptide molecular weight markers andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these markers, depending upon theextent of modification. The size of these polypeptide molecular weightmarkers can be most heterogeneous with fragments of polypeptide derivedfrom the extracellular portion of the polypeptide. Consistent molecularweight markers can be obtained by using polypeptides derived entirelyfrom the transmembrane and cytoplasmic regions, pretreating withN-glycanase to remove glycosylation, or expressing the polypeptides inbacterial hosts.

As stated above, the invention provides isolated and purifiedpolypeptides, both recombinant and non-recombinant. Variants andderivatives of native polypeptides can be obtained by mutations ofnucleotide sequences coding for native polypeptides. Alterations of thenative amino acid sequence can be accomplished by any of a number ofconventional methods. Mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,all of which are incorporated by reference.

Polypeptides of the invention can be modified to create polypeptidederivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, polyethylene glycol (PEG)groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of polypeptides of the invention can be prepared by linkingthe chemical moieties to functional groups on polypeptide amino acidside chains or at the N-terminus or C-terminus of a polypeptide of theinvention or the extracellular domain thereof. Other derivatives ofpolypeptides within the scope of this invention include covalent oraggregative conjugates of these polypeptides or peptide fragments withother proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatecan comprise a signal or leader polypeptide sequence (e.g. the α-factorleader of Saccharomyces) at the N-terminus of a polypeptide of theinvention. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

Polypeptide conjugates can comprise peptides added to facilitatepurification and identification of polypeptides of the invention. Suchpeptides include, for example, poly-His or the antigenic identificationpeptides described in U.S. Pat. No. 5,011,912 and in Hopp et al.,Bio/Technology 6:1204, 1988.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Glycosyl groups can be removed through conventional methods,in particular those utilizing glycopeptidase. In general, glycosylatedpolypeptides of the invention can be incubated with a molar excess ofglycopeptidase (Boehringer Mannheim).

Correspondingly, equivalent DNA constructs that encode various additionsor substitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Known proceduresfor inactivating N-glycosylation sites in proteins include thosedescribed in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporatedby reference.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding, or substituting residues to alter Arg—Arg, Arg-Lys,and Lys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys—Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys—Lys represents aconservative and preferred approach to inactivating KEX2 sites.

The invention further encompasses isolated fragments andoligonucleotides derived from the nucleotide sequence of SEQ ID NO:1, 2,3, 4, 5, 6, 13 or 15. Nucleic acid sequences within the scope of theinvention include isolated DNA and RNA sequences that hybridize to thenative nucleotide sequences disclosed herein under conditions ofmoderate or severe stringency, and which encode polypeptides orfragments thereof of the invention. These isolated DNA and RNA sequencesalso include full length DNA or RNA molecules encoding for polypeptideswith kinase activity. As used herein, conditions of moderate stringency,as known to those having ordinary skill in the art, and as defined bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), include useof a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6×SSCat 42° C. (or other similar hybridization solution, such as Stark'ssolution, in 50% formamide at 42° C.), and washing conditions of about60° C., 0.5×SSC, 0.1% SDS. Conditions of high stringency are defined ashybridization conditions as above, and with washing at 68° C., 0.2×SSC,0.1% SDS. The skilled artisan will recognize that the temperature andwash solution salt concentration can be adjusted as necessary accordingto factors such as the length of the probe.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NO:1, 2, 3, 4, 5, 6, 13 or 15, and still encode apolypeptide having the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11,12, 14 or 16. Such variant DNA sequences can result from silentmutations (e.g., occurring during PCR amplification), or can be theproduct of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA derived from thecoding region of a native mammalian gene; (b) cDNA comprising thenucleotide sequence of SEQ ID NO: 1, 2, 3,4, 5, 6, 13 or 15; (c) DNAencoding the polypeptides of SEQ ID NO:7, 8,9, 10, 11, 12, 14 or 16; (d)DNA capable of hybridization to a DNA of (a) under conditions ofmoderate stringency and which encodes polypeptides of the invention; and(e) DNA which is degenerate as a result of the genetic code to a DNAdefined in (a), (b), (c), or (d) and which encodes polypeptides of theinvention. Of course, polypeptides encoded by such DNA equivalentsequences are encompassed by the invention.

DNA that is equivalent to the DNA sequence of SEQ ID NO:1, 2, 3, 4, 5,6, 13 or 15 will hybridize under moderately stringent conditions to thedouble-stranded native DNA sequence that encode polypeptides comprisingamino acid sequences of SEQ ID NO:7, 8, 9, 10, 11, 12, 14 or 16.Examples of polypeptides encoded by such DNA, include, but are notlimited to, polypeptide fragments and polypeptides comprisinginactivated N-glycosylation site(s), inactivated protease processingsite(s), or conservative amino acid substitution(s), as described above.Polypeptides encoded by DNA derived from other mammalian species,wherein the DNA will hybridize to the complement of the DNA of SEQ IDNO:1, 2, 3, 4, 5, 6, 13 or 15 are also encompassed.

Recombinant expression vectors containing a nucleic acid sequenceencoding polypeptides of the invention can be prepared using well knownmethods. The expression vectors include a DNA sequence of the inventionoperably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe DNA sequence of the invention. Thus, a promoter nucleotide sequenceis operably linked to a DNA sequence if the promoter nucleotide sequencecontrols the transcription of the DNA sequence of the invention. Theability to replicate in the desired host cells, usually conferred by anorigin of replication, and a selection gene by which transformants areidentified can additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with polypeptides of the invention can beincorporated into expression vectors. For example, a DNA sequence for asignal peptide (secretory leader) can be fused in-frame to thenucleotide sequence of the invention so that the polypeptide isinitially translated as a fusion protein comprising the signal peptide.A signal peptide that is functional in the intended host cells enhancesextracellular secretion of the polypeptide. The signal peptide can becleaved from the polypeptide upon secretion of polypeptide from thecell.

Suitable host cells for expression of polypeptides of the inventioninclude prokaryotes, yeast or higher eukaryotic cells. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described, for example, in Pouwels etal. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985).Cell-free translation systems could also be employed to producepolypeptides of the invention using RNAs derived from DNA constructsdisclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a polypeptide of the invention can include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metcan be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and a DNA sequence of the invention are insertedinto the pBR322 vector. Other commercially available vectors include,for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) andPGEM1 (Promega Biotec, Madison, Wis., USA). Other commercially availablevectors include those that are specifically designed for the expressionof proteins; these would include pMAL-p2 and pMAL-c2 vectors that areused for the expression of proteins fused to maltose binding protein(New England Biolabs, Beverly, Mass., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include P-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λP_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection, which incorporate derivatives of the λP_(L)promoter, include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

DNA of the invention can be cloned in-frame into the multiple cloningsite of an ordinary bacterial expression vector. Ideally the vectorcontains an inducible promoter upstream of the cloning site, such thataddition of an inducer leads to high-level production of the recombinantprotein at a time of the investigator's choosing. For some proteins,expression levels can be boosted by incorporation of codons encoding afusion partner (such as hexahistidine) between the promoter and the geneof interest. The resulting “expression plasmid” can be propagated in avariety of prokaryotic hosts such as E. coli.

For expression of the recombinant protein, the bacterial cells arepropagated in growth medium until reaching a pre-determined opticaldensity. Expression of the recombinant protein is then induced, e.g. byaddition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activatesexpression of proteins from plasmids containing a lac operator/promoter.After induction (typically for 1-4 hours), the cells are harvested bypelleting in a centrifuge, e.g. at 5,000×G for 20 minutes at 4° C.

For recovery of the expressed protein, the pelleted cells may beresuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and thenpassed two or three times through a French press. Most highly-expressedrecombinant proteins form insoluble aggregates known as inclusionbodies. Inclusion bodies can be purified away from the soluble proteinsby pelleting in a centrifuge at 5,000×G for 20 minutes, 4° C. Theinclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% TritonX-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT.Any material that cannot be dissolved is removed by centrifugation(10,000×G for 20 minutes, 20° C.). The protein of interest will, in mostcases, be the most abundant protein in the resulting clarifiedsupernatant. This protein may be “refolded” into the active conformationby dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl₂/5 mM Zn(OAc)₂/1 mMGSSG/0.1 mM GSH. After refolding, purification can be carried out by avariety of chromatographic methods such as ion exchange or gelfiltration. In some protocols, initial purification may be carried outbefore refolding. As an example, hexahistidine-tagged fusion proteinsmay be partially purified on immobilized nickel.

While the preceding purification and refolding procedure assumes thatthe protein is best recovered from inclusion bodies, those skilled inthe art of protein purification will appreciate that many recombinantproteins are best purified out of the soluble fraction of cell lysates.In these cases, refolding is often not required, and purification bystandard chromatographic methods can be carried out directly.

Polypeptides of the invention alternatively can be expressed in yeasthost cells, preferably from the Saccharomyces genus (e.g., S.cerevisiae). Other genera of yeast, such as Pichia, K. lactis, orKluyveromyces, can also be employed. Yeast vectors will often contain anorigin of replication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980), or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et al., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli can be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp gene and origin of replication) into the above-describedyeast vectors.

The yeast α-factor leader sequence can be employed to direct secretionof a polypeptide of the invention. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.Other leader sequences suitable for facilitating secretion ofrecombinant polypeptides from yeast hosts are known to those of skill inthe art. A leader sequence can be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine, and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence can be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant polypeptides of the invention. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also can be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line (ATCC CRL10478) derived from the African green monkey kidney cell line CVI (ATCCCCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used totransfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417, 1987). In addition, electroporation can be used totransfect mammalian cells using conventional procedures, such as thosein Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol.1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stabletransformants can be performed using methods known in the art, such as,for example, resistance to cytotoxic drugs. Kaufman et al., Meth. inEnzymology 185:487-511, 1990, describes several selection schemes, suchas dihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B 11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acadi Sci. USA 77:42164220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B 11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534) and thetripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras etal., J. Biol. Chem. 257:13475-13491, 1982). The internal ribosome entrysite (IRES) sequences of viral origin allows dicistronic mRNAs to betranslated efficiently (Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295-300, 1993; Ramesh et al., Nucleic Acids Research24:2697-2700, 1996). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman, Meth. in Enzymology,1990). Exemplary expression vectors that employ dicistronic mRNAs arepTR-DC/GFP described by Mosser et al., Biotechniques 22:150-161, 1997,and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp.529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335-348, 1989. Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are describedin EP-A0367566, and in U.S. patent application Ser. No. 07/701,415,filed May 16, 1991, incorporated by reference herein. The vectors can bederived from retroviruses. In place of the native signal sequence, aheterologous signal sequence can be added, such as the signal sequencefor IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence forIL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL4signal peptide described in EP 367,566; the type I IL-1 receptor signalpeptide described in U.S. Pat. No. 4,968,607; and the type II IL-1receptor signal peptide described in EP 460,846. Another usefulexpression vector, pFLAG, can be used. FLAG technology is centered onthe fusion of a low molecular weight (1 kD), hydrophilic, FLAG markerpeptide to the N-Terminus of a recombinants protein expressed by thepFLAG-1™ Expression Vector (1) (obtained from IBI Kodak).

An isolated and purified polypeptide according to the invention can beproduced by recombinant expression systems as described above orpurified from naturally occurring cells. Polypeptides can besubstantially purified, as indicated by a single protein band uponanalysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

One process for producing polypeptides of the invention comprisesculturing a host cell transformed with an expression vector comprising aDNA sequence that encodes a polypeptide of the invention underconditions sufficient to promote expression of the polypeptide. Thepolypeptide is then recovered from culture medium or cell extracts,depending upon the expression system employed. As is known to theskilled artisan, procedures for purifying a recombinant protein willvary according to such factors as the type of host cells employed andwhether or not the recombinant protein is secreted into the culturemedium. For example, when expression systems that secrete therecombinant protein are employed, the culture medium first can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred. Finally, one or more reversed-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media, (e.g., silica gel having pendant methyl or otheraliphatic groups) can be employed to further purify the polypeptides.Some or all of the foregoing purification steps, in variouscombinations, are well known and can be employed to provide an isolatedand purified recombinant protein.

It is possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention, can be bound to asolid phase such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cel. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

In the methods described above, suitable polypeptide-binding proteinsare anti-polypeptide antibodies, and other proteins that are capable ofhigh-affinity binding of polypeptides of the invention. A preferredpolypeptide-binding protein is an polypeptide monoclonal antibody.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to expresspolypeptides of the invention as a secreted polypeptide in order tosimplify purification. Secreted recombinant polypeptide from a yeasthost cell fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al.describe two sequential, reversed-phase HPLC steps for purification ofrecombinant human IL-2 on a preparative HPLC column.

In yet another embodiment of the invention, antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to a target mRNA sequence(forming a duplex) or to the sequence in the double-stranded DNA helix(forming a triple helix) can be made according to the invention.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of cDNA (SEQ ID NO:1, 2, 3, 4,5, 6, 13 or 15). Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to about 30 nucleotides. Theability to create an antisense or a sense oligonucleotide, based upon acDNA sequence for a given protein is described in, for example, Steinand Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of complexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus canbe used to block expression of polypeptides of the invention. Antisenseor sense oligonucleotides further comprise oligonucleotides havingmodified sugar-phosphodiester backbones (or other sugar linkages, suchas those described in WO 91/06629) and wherein such sugar linkages areresistant to endogenous nucleases. Such oligonucleotides with resistantsugar linkages are stable in vivo (i.e., capable of resisting enzymaticdegradation), but retain sequence specificity to be able to bind totarget nucleotide sequences. Other examples of sense or antisenseoligonucleotides include those oligonucleotides that are covalentlylinked to organic moieties, such as those described in WO 90/10448, andother moieties that increase affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents, such as ellipticine, and alkylating agents ormetal complexes can be attached to sense or antisense oligonucleotidesto modify binding specificities of the antisense or senseoligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides can be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application U.S. Ser. No.90/02656).

Alternatively, sense or antisense oligonucleotides also can beintroduced into a cell containing the target nucleotide sequence byformation of a conjugate with a ligand binding molecule, as described inWO 91/04753. Suitable ligand binding molecules include, but are notlimited to, cell surface receptors, growth factors, other cytokines, orother ligands that bind to cell surface receptors. Preferably,conjugation of the ligand binding molecule does not substantiallyinterfere with the ability of the ligand binding molecule to bind to itscorresponding molecule or receptor, or block entry of the sense orantisense oligonucleotide or its conjugated version into the cell.

In yet another embodiment, a sense or an antisense oligonucleotide canbe introduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

Another embodiment of the invention relates to therapeutic uses ofkinases. Kinases play a central role in cellular signal transduction. Assuch, alterations in kinase expression and/or activation can haveprofound effects on a plethora of cellular processes, including, but notlimited to, activation or inhibition of cell specific responses,proliferation, and programmed cell death (apoptosis). Over expression ofcloned kinases or of catalytically inactive mutants of kinases has beenused to identify the role a particular kinase plays in mediatingspecific signaling events.

Kinase mediated cellular signaling often involves a molecular activationcascade, during which an activated kinase propagates a ligand-receptormediated signal by specifically phosphorylating target substrates. Thesesubstrates can themselves be kinases which become activated followingphosphorylation. Alternatively, they can be adaptor molecules thatfacilitate down stream signaling through protein-protein interactionfollowing phosphorylation. Regardless of the nature of the substratemolecule(s), expressed catalytically active versions of the putativekinases in the invention can be used to identify what substrate(s) wererecognized and phosphorylated by the kinase(s) of the invention. Assuch, these kinases can be used as reagents to identify novel moleculesinvolved in signal transduction pathways.

Knowledge of a particular kinase would enable one to enzymatically labelthe substrate with ³²P thereby facilitating identification and also touse the resultant radiolabeled protein or a peptide derived from acertain region of the protein as a substrate probe to identify andisolate specific phosphatases. Phosphatases are enzymes whose functionis to remove phosphates from selected phosphoproteins; they perform thereverse function of kinases.

In some systems specific glycosylations with N-acetyl glucosamineresidues have been described as a biological counter balance to kinases.That is, glycosidases have been suggested to compete with kinases forthe same serine or threonine residues to covalently modify. Therefore,the kinase can be used to dissect dynamic interactions between kinaseand phosphatase, and also between kinase and glycosidase, and finally toexamine the dynamic interactions among kinase, phosphatase andglycosidase together.

Kinases phosphorylate target serine, threonine or tyrosine residues inthe context of specific recognition motifs. Recognition motifs canconsist solely of primary structure or in some cases recognitionrequires more complex structural features. One can take advantage ofkinases with strict primary sequence recognition requirements by usingthem as a general labeling reagent. Nucleotides coding for the aminoacids recognized by a particular kinase could be engineered onto eitherend of a protein on interest, thereby “tagging” the molecule. Theexpressed, tagged protein could be ³²P-labeled at a known site on theengineered tag by its specific kinase, thus generating a well defined,radiolabeled protein.

Because kinases are phosphotransferases, they must take part inprotein-protein interactions with at least one or more substratemolecules, i.e. its phosphate recipient(s). Therefore, kinases orpolypeptides comprised of portions of a kinase could be used as “baits”in the yeast two hybrid system by well established molecular biologytechniques, to identify molecules that interact directly with thepolypeptide.

Alternatively, polypeptides of the invention could be engineered priorto expression with a tag such as poly-His or FLAG, then be expressed andpurified using either nickel chelate chromatography or anti-FLAGantibody coupled to a resin, respectively. Once bound to the resin, thepolypeptide of the invention could be covalently attached using abifunctional cross-linking agent using well established techniques. Thecovalently bound polypeptide to the resin could then be used to purifymolecules from cell lysates or cell supernatants (following treatmentwith various reagent) through their affinity for the polypeptide of theinvention.

Isolated and purified kinase polypeptides or a fragment thereof of theinvention can also be useful as a therapeutic agent in inhibitingsignaling. Polypeptides are introduced into the intracellularenvironment by well-known means, such as by encasing the protein inliposomes or coupling it to a monoclonal antibody targeted to a specificcell type.

DNA, polypeptides, and antibodies against polypeptides of the inventioncan be used as reagents in a variety of research protocols. A sample ofsuch research protocols are given in Sambrook et al. Molecular Cloning:A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor LaboratoryPress, (1989). For example, these reagents can serve as markers for cellspecific or tissue specific expression of RNA or proteins. Similarly,these reagents can be used to investigate constitutive and transientexpression of RNA or polypeptides. The DNA can be used to determine thechromosomal location of DNA and to map genes in relation to thischromosomal location. The DNA can also be used to examine geneticheterogeneity and heredity through the use of techniques such as geneticfingerprinting, as well as to identify risks associated with geneticdisorders. The DNA can be further used to identify additional genesrelated to the DNA and to establish evolutionary trees based on thecomparison of sequences. The DNA and polypeptides can be used to selectfor those genes or proteins that are homologous to the DNA orpolypeptides, through positive screening procedures such as Southernblotting and immunoblotting and through negative screening proceduressuch as subtraction.

The polypeptides and fragments of the invention can also be used as areagent to identify (a) any protein that polypeptide regulates, and (b)other proteins with which it might interact. Polypeptides could be usedby coupling recombinant protein to an affinity matrix, or by using themas a bait in the 2-hybrid system. The polypeptides and fragments thereofcan be used as reagents in the study of the kinase signaling pathway asa reagent to block kinase signaling.

A hallmark of protein kinases is their ability to phosphorylate otherproteins and to auto-phosphorylate. Therefore, in one aspect of theinvention, the isolated polypeptides with kinase activity can be used inassays to phosphorylate target proteins, radiolabel target proteins with³²P, and identify proteins having phosphatase activity. Exemplarymethods of phosphorylation assays set forth above are disclosed in U.S.Pat. No. 5,447,860 which is incorporated herein by reference. Inaddition to full length polypeptides, the invention also includes theisolated active kinase domains of kinases, such as the intracellulardomain of transmembrane bound kinases and the cytoplasmic kinases, whichcan function as reagents in kinase assays. Further, soluble forms of theextracellular domains of the kinases are useful in inhibiting thenatural ligand-receptor interaction.

When used as a therapeutic agent, polypeptides of the invention can beformulated into pharmaceutical compositions according to known methods.The polypeptides can be combined in admixture, either as the sole activematerial or with other known active materials, with pharmaceuticallysuitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,adjuvants and/or carriers. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Co. In addition, such compositions can contain thepolypeptides complexed with polyethylene glycol (PEG), metal ions, orincorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Such compositions will influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance of polypeptides of the invention.

The dosage of the composition can be readily determined by those ofordinary skill in the art. The amount to be administered and thefrequency of administration can be determined empirically and will takeinto consideration the age and size of the patient being treated, aswell as the malady being treated.

Treatment comprises administering the composition by any method familiarto those of ordinary skill in the art, including intravenous,intraperitoneal, intracorporeal injection, intra-articular,intraventricular, intrathecal, intramuscular, subcutaneous, topically,tonsillar, intranasally, intravaginally, and orally. The composition mayalso be given locally, such as by injection into the particular area,either intramuscularly or subcutaneously.

Within the therapeutic and research aspects of the invention,polypeptides of the invention, and peptides based on the amino acidsequence thereof, can be utilized to prepare antibodies thatspecifically bind to the polypeptides. The term “antibodies” is meant toinclude polyclonal antibodies, monoclonal antibodies, fragments thereofsuch as F(ab′)2, and Fab fragments, as well as any recombinantlyproduced binding partners. Antibodies are defined to be specificallybinding if they bind polypeptides of the invention with a K_(a) ofgreater than or equal to about 10⁷ M⁻¹. Affinities of binding partnersor antibodies can be readily determined using conventional techniques,for example those described by Scatchard et al., Ann. N.Y Acad. Sci.,51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well-known in the art.In general, purified polypeptides of the invention, or a peptide basedon the amino acid sequence of polypeptides of the invention that isappropriately conjugated, is administered to the host animal typicallythrough parenteral injection. The immunogenicity of these polypeptidescan be enhanced through the use of an adjuvant, for example, Freund'scomplete or incomplete adjuvant. Following booster immunizations, smallsamples of serum are collected and tested for reactivity to thepolypeptides. Examples of various assays useful for such determinationinclude those described in: Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well asprocedures such as countercurrent immuno-electrophoresis (CIEP),radioimmunoassay, radio-immunoprecipitation, enzyme-linkedimmuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, seeU.S. Pat. Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies can be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.32,011, 4,902,614, 4,543,439, and 4,411,993; RE Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980. Briefly, the host animals,such as Balb/c mice are injected intraperitoneally at least once, andpreferably at least twice at about 3 week intervals with isolated andpurified polypeptides or conjugated polypeptides of the invention,optionally in the presence of adjuvant. 10 μg of isolated and purifiedpolypeptide of the invention or peptides based on the amino acidsequence of polypeptides of the invention in the presence of RIBIadjuvant (RIBI Corp., Hamilton, Mont.). Mouse sera are then assayed byconventional dot blot technique or antibody capture (ABC) to determinewhich animal produces the highest level of antibody and whose spleencells are the best candidate for fusion. Approximately two to threeweeks later, the mice are given an intravenous boost of the polypeptidesor conjugated polypeptides such as 3 μg suspended in sterile PBS. Miceare later sacrificed and spleen cells fused with commercially availablemyeloma cells, such as Ag8.653 (ATCC), following established protocols.Briefly, the myeloma cells are washed several times in media and fusedto mouse spleen cells at a ratio of about three spleen cells to onemyeloma cell. The fusing agent can be any suitable agent used in theart, for example, polyethylene glycol (PEG) or more preferably, 50% PEG:10% DMSO (Sigma). Fusion is plated out into, for example, twenty 96-wellflat bottom plates (Corning) containing an appropriate medium, such asHAT supplemented DMEM media and allowed to grow for eight days.Supernatants from resultant hybridomas are collected and added to, forexample, a 96-well plate for 60 minutes that is first coated with goatanti-mouse Ig. Following washes, ¹²⁵I-polypeptide or peptides of theinvention are added to each well, incubated for 60 minutes at roomtemperature, and washed four times. Positive wells can be subsequentlydetected by conventional methods, such as autoradiography at −70° C.using Kodak X-Omat S film. Positive clones can be grown in bulk cultureand supernatants are subsequently purified, such as over a Protein Acolumn (Pharmacia). It is understood of course that many techniquescould be used to generate antibodies against polypeptides and fragmentedpeptides of the invention and that this embodiment in no way limits thescope of the invention.

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7:394 (1989).

Other types of “antibodies” can be produced using the informationprovided herein in conjunction with the state of knowledge in the art.For example, antibodies that have been engineered to contain elements ofhuman antibodies that are capable of specifically binding polypeptidesof the invention are also encompassed by the invention.

Once isolated and purified, the antibodies against polypeptides of theinvention can be used to detect the presence of the polypeptides in asample using established assay protocols. Further, the antibodies of theinvention can be used therapeutically or for research purposes to bindto the polypeptides and inhibit its activity in vivo or in vitro.

Antibodies immunoreactive with polypeptides of the invention, and inparticular, monoclonal antibodies against these polypeptides, are nowmade available through the invention. Such antibodies can be useful forinhibiting polypeptide activity in vivo and for detecting the presenceof polypeptides of the invention in a sample.

In another embodiment, antibodies generated against a polypeptide andfragmented peptides of the invention can be used in combination withpolypeptide or fragmented peptide molecular weight markers of theinvention to enhance the accuracy in the use of these molecular weightmarkers to determine the apparent molecular weight and isoelectric pointof a sample protein. Polypeptide or fragmented peptide molecular weightmarkers of the invention can be mixed with a molar excess of a sampleprotein and the mixture can be resolved by two dimensionalelectrophoresis by conventional means. Polypeptides can be transferredto a suitable protein binding membrane, such as nitrocellulose, byconventional means and detected by the antibodies of the invention.

The purified polypeptides according to the invention will facilitate thediscovery of inhibitors of such polypeptides. The use of a purifiedpolypeptide of the invention in the screening of potential inhibitorsthereof is important and can eliminate or reduce the possibility ofinterfering reactions with contaminants.

In addition, polypeptides of the invention can be used forstructure-based design of polypeptide-inhibitors. Such structure-baseddesign is also known as “rational drug design.” The polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of the polypeptide structural information inmolecular modeling software systems to assist in inhibitor design andinhibitor- polypeptide interaction is also encompassed by the invention.Such computer-assisted modeling and drug design can utilize informationsuch as chemical conformational analysis, electrostatic potential of themolecules, protein folding, etc. For example, most of the design ofclass-specific inhibitors of metalloproteases has focused on attempts tochelate or bind the catalytic zinc atom. Synthetic inhibitors areusually designed to contain a negatively-charged moiety to which isattached a series of other groups designed to fit the specificitypockets of the particular protease. A particular method of the inventioncomprises analyzing the three dimensional structure of polypeptides ofthe invention for likely binding sites of substrates, synthesizing a newmolecule that incorporates a predictive reactive site, and assaying thenew molecule as described above.

The polypeptides of the present invention may also be used in ascreening assay to identify compounds and small molecules which inhibit(antagonize) or enhance (agonize) activation of the polypeptides of theinstant invention. Thus, for example, polypeptides of the invention maybe used to identify antagonists and agonists from cells, cell-freepreparations, chemical libraries, and natural product mixtures. Theantagonists and agonists may be natural or modified substrates, ligands,enzymes, receptors, etc. of the polypeptides of the instant invention,or may be structural or functional mimetics of the polypeptides.Potential antagonists of the polypeptides of the instant invention mayinclude small molecules, peptides, and antibodies that bind to andoccupy a binding site of the polypeptides, causing them to beunavailable to bind to their ligands and therefore preventing normalbiological activity. Other potential antagonists are antisense moleculeswhich may hybridize to mRNA in vivo and block translation of the mRNAinto the polypeptides of the instant invention. Potential agonistsinclude small molecules, peptides and antibodies which bind to theinstant polypeptides and elicit the same or enhanced biological effectsas those caused by the binding of the polypeptides of the instantinvention.

Small molecule agonists and antagonists are usually less than 10 Kmolecular weight and may possess a number of physiochemical andpharmacological properties that enhance cell penetration, resistdegradation and prolong their physiological half-lives. (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994).)Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, may be used to bind to and inhibit thepolypeptides of the instant invention by blocking the commencement of asignaling cascade. It is preferable that the antibodies are humanized,and more preferable that the antibodies are human. The antibodies of thepresent invention may be prepared by any of a variety of well-knownmethods.

Specific screening methods are known in the art and many are extensivelyincorporated in high throughput test systems so that large numbers oftest compounds can be screened within a short amount of time. The assayscan be performed in a variety of formats, including protein-proteinbinding assays, biochemical screening assays, immunoassays, cell basedassays, etc. These assay formats are well known in the art. Thescreening assays of the present invention are amenable to screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides and other antagonists andagonists.

One embodiment of a method for identifying molecules which antagonize orinhibit the polypeptides involves adding a candidate molecule to amedium which contains cells that express the polypeptides of the instantinvention; changing the conditions of said medium so that, but for thepresence of the candidate molecule, the polypeptides would be bound totheir ligands; and observing the binding and stimulation or inhibitionof a functional response. The activity of the cells which were contactedwith the candidate molecule may then be compared with the identicalcells which were not contacted and agonists and antagonists of thepolypeptides of the instant invention may be identified. The measurementof biological activity may be performed by a number of well-knownmethods such as measuring the amount of protein present (e.g. an ELISA)or of the protein's activity. A decrease in biological stimulation oractivation would indicate an antagonist. An increase would indicate anagonist. Specifically, one embodiment of the instant invention includesagonists and antagonists of QQ335 1, SS1771, SS1771A and truncatedQQ3351.

Screening assays can further be designed to find molecules that mimicthe biological activity of the polypeptides of the instant invention.Molecules which mimic the biological activity of a polypeptide may beuseful for enhancing the biological activity of the polypeptide. Toidentify compounds for therapeutically active agents that mimic thebiological activity of a polypeptide, it must first be determinedwhether a candidate molecule binds to the polypeptide. A bindingcandidate molecule is then added to a biological assay to determine itsbiological effects. The biological effects of the candidate molecule arethen compared to the those of the polypeptide.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan recognizes many other embodiments are encompassed by theclaimed invention.

16 1 181 DNA Homo sapiens 1 gtacgccatg aaggtgctgc gcaaggcggc gctggtgcagcgcgccaaga cgcaagagca 60 cacgcgcacc gagcgctcgg tgctggagct ggtgcgccaggcgcccttcc tggtcacgct 120 gcactacgct ttccagacgg atgccaagct gcacctcatcctggactatg tgagcggcgg 180 g 181 2 221 DNA Homo sapiens 2 cccgagaggtgccacatcag accgcctccg acttcgtgcg ggactcggcg gccagccacc 60 aggcggagcccgaggcgtac gagcggcgcg tgtgcttcct gcttctgcaa ctctgcaacg 120 ggctggagcacctgaaggag cacgggatca tccaccggga cctgtgcctg gagaacctgc 180 tgctggtgcactgcaccctc caggccggcc ccgggcccgc c 221 3 1085 DNA Homo sapiens 3cgggcagggc tggagctggg ctgggatccc gagctcggca gcagcgcagc gggccggccc 60acctgctggt gccctggagg ctctgagccc cggcggcgcc cgggcccacg cggaacgacg 120gggcgagatg cgagccaccc ctctggctgc tcctgcgggt tccctgtcca ggaagaagcg 180gttggagttg gatgacaact tagataccga gcgtcccgtc cagaaacgag ctcgaagtgg 240gccccagccc agactgcccc cctgcctgtt gcccctgagc ccacctactg ctccagatcg 300tgcaactgct gtggccactg cctcccgtct tgggccctat gtcctcctgg agcccgagga 360gggcgggcgg gcctaccagg ccctgcactg ccctacaggc actgagtata cctgcaaggt 420gtaccccgtc caggaagccc tggccgtgct ggagccctac gcgcggctgc ccccgcacaa 480gcatgtggct cggcccactg aggtcctggc tggtacccag ctcctctacg cctttttcac 540tcggacccat ggggacatgc acagcctggt gcgaagccgc caccgtatcc ctgagcctga 600ggctgccgtg ctcttccgcc agatggccac cgccctggcg cactgtcacc agcacggtct 660ggtcctgcgt gatctcaagc tgtgtcgctt tgtcttcgct gaccgtgaga ggaagaagct 720ggtgctggag aacctggagg actcctgcgt gctgactggg ccagatgatt ccctgtggga 780caagcacgcg tgcccagcct acgtgggacc tgagatactc agctcacggg cctcatactc 840gggcaaggca gccgatgtct ggagcctggg cgtggcgctc ttcaccatgc tggccggcca 900ctaccccttc caggactcgg agcctgtcct gctcttcggc aagatccgcc gcggggccta 960cgccttgcct gcaggcctct cggcccctgc ccgctgtctg gttcgctgcc tccttcgtcg 1020ggagccagct gaacggctca cagccacagg catcctcctg cacccctggc tgcgacagga 1080cccga 1085 4 388 DNA Homo sapiens 4 cagcgagaag ccgacatgca tcgcctcttcaatcacccca acatccttcg cctcgtggct 60 tactgtctga gggaacgggg tgctaagcatgaggcctggc tgctgctacc attcttcaag 120 agaggtacgc tgtggaatga gatagaaaggctgaaggaca aaggcaactt cctgaccgag 180 gatcaaatcc tttggctgct gctggggatctgcagaggcc ttgaggccat tcatgccaag 240 ggttatgcct acagagactt gaagcccaccaatatattgc ttggagatga ggggcagcca 300 gttttaatgg acttgggttc catgaatcaagcatgcatcc atgtggaggg ctcccgccag 360 gctctgaccc tgcaggactg ggcagccc 3885 1555 DNA Homo sapiens 5 atgctaacta gtttaaacag atcttggaac gagacgacctgctgtggaag agcgagcttt 60 ttggaactgt gcacgggaca gattggacgc acacccctcgggaggcgcga aggcatggaa 120 aatttgaagc atattatcac ccttggccag gtcatccacaaacggtgtga agagatgaaa 180 tactgcaaga aacagtgccg gcgcctgggc caccgcgtcctcggcctgat caagcctctg 240 gagatgctcc aggaccaagg aaagaggagc gtgccctctgagaagttaac cacagccatg 300 aaccgcttca aggctgccct ggaggaggct aatggggagatagaaaagtt cagcaataga 360 tccaatatct gcaggtttct aacagcaagc caggacaaaatactcttcaa ggacgtgaac 420 aggaagctga gtgatgtctg gaaggagctc tcgctgttacttcaggttga gcaacgcatg 480 cctgtttcac ccataagcca aggagcgtcc tgggcacaggaagatcagca ggatgcagac 540 gaagacaggc gagctttcca gatgctaaga agagataatgaaaaaataga agcttcactg 600 agacgattag aaatcaacat gaaagaaatc aaggaaactttgaggcagta tttaccacca 660 aaatgcatgc aggagatccc gcaagagcaa atcaaggagatcaagaagga gcagctttca 720 ggatccccgt ggattctgct aagggaaaat gaagtcagcacactttataa aggagaatac 780 cacagagctc cagtggccat aaaagtattc aaaaaactccaggctggcag cattgcaata 840 gtgaggcaga ctttcaataa ggagatcaaa accatgaagaaattcgaatc tcccaacatc 900 ctgcgtatat ttgggatttg cattgatgaa acagtgactccgcctcaatt ctccattgtc 960 atggagtact gtgaactcgg gaccctgagg gagctgttggatagggaaaa agacctcaca 1020 cttggcaagc gcatggtcct agtcctgggg gcagcccgaggcctataccg gctacaccat 1080 tcagaagcac ctgaactcca cggaaaaatc agaagctcaaacttcctggt aactcaaggc 1140 taccaagtga agcttgcagg atttgagttg aggaaaacacagacttccat gagtttggga 1200 actacgagag aaaagacaga cagagtcaaa tctacagcatatctctcacc tcaggaactg 1260 gaagatgtat tttatcaata tgatgtaaag tctgaaatatacagctttgg aatcgtcctc 1320 tgggaaatcg ccactggaga tatcccgttt caaggctgtaattctgagaa gatccgcaag 1380 ctggtggctg tgaagcggca gcaggagcca ctgggtgaagactgcccttc agagctgcgg 1440 gagatcattg atgagtgccg ggcagcaggt cgtctcgttccaagatctgt agcggccgcc 1500 cgggccgtcg acgtttaaac gcgtggccct cgagaggttttccgatccgg tcgat 1555 6 1498 DNA Homo sapiens 6 cttcccgctg gacgtggagtacggaggccc agaccggagg tgcccgcctc cgccctaccc 60 gaagcacctg ctgctgcgcagcaagtcgga gcagtacgac ctggacagcc tgtgcgcagg 120 catggagcag agcctccgtgcgggccccaa cgagcccgag ggcggcgaca agagccgcaa 180 aagcgccaag ggggacaaaggcggaaagga taaaaagcag attcagacct ctcccgttcc 240 cgtccgcaaa aacagcagagacgaagagaa gagagagtca cgcatcaaga gctactcgcc 300 atacgccttt aagttcttcatggagcagca cgtggagaat gtcatcaaaa cctaccagca 360 gaaggttaac cggaggctgcagctggagca agaaatggcc aaagctggac tctgtgaagc 420 tgagcaggag cagatgcggaagatcctcta ccagaaagag tctaattaca acaggttaaa 480 gagggccaag atggacaagtctatgtttgt caagatcaaa accctgggga tcggtgcctt 540 tggagaagtg tgccttgcttgtaaggtgga cactcacgcc ctgtacgcca tgaagaccct 600 aaggaaaaag gatgtcctgaaccggaatca ggtggcccac gtcaaggccg agagggacat 660 cctggccgag gcagacaatgagtgggtggt caaactctac tactccttcc aagacaaaga 720 cagcctgtac tttgtgatggactacatccc tggtggggac atgatgagcc tgctgatccg 780 gatggaggtc ttccctgagcacctggcccg gttctacatc gcagagctga ctttggccat 840 tgagagtgtc cacaagatgggcttcatcca ccgagacatc aagcctgata acattttgat 900 agatctggat ggtcacattaaactcacaga tttcggcctc tgcactgggt tcaggtggac 960 tcacaattcc aaatattaccagaaagggag ccatgtcaga caggacagca tggagcccag 1020 cgacctctgg gatgatgtgtctaactgtcg gtgtggggac aggctgaaga ccctagagca 1080 gagggcgcgg aagcagcaccagaggtgcct ggcacattca ctggtgggga ctccaaacta 1140 catcgcaccc gaggtgctcctccgcaaagg gtacactcaa ctctgtgact ggtggagtgt 1200 tggagtgatt ctcttcgagatgctggtggg gcagccgccc tttttggcac ctactcccac 1260 agaaacccag ctgaaggtgatcaactggga gaacacgctc cacattccag cccaggtgaa 1320 gctgagccct gaggccagggacctcatcac caagctgtgc tgctccgcag accaccgcct 1380 ggggcggaat ggggccgatgacctgaaggc ccaccccttc ttcagcgcca ttgacttctc 1440 cagtgacatc cggaagcatccagcccccta cgttcccacc atcagccacc ccatggag 1498 7 60 PRT Homo sapiens 7Tyr Ala Met Lys Val Leu Arg Lys Ala Ala Leu Val Gln Arg Ala Lys 1 5 1015 Thr Gln Glu His Thr Arg Thr Glu Arg Ser Val Leu Glu Leu Val Arg 20 2530 Gln Ala Pro Phe Leu Val Thr Leu His Tyr Ala Phe Gln Thr Asp Ala 35 4045 Lys Leu His Leu Ile Leu Asp Tyr Val Ser Gly Gly 50 55 60 8 73 PRTHomo sapiens 8 Arg Glu Val Pro His Gln Thr Ala Ser Asp Phe Val Arg AspSer Ala 1 5 10 15 Ala Ser His Gln Ala Glu Pro Glu Ala Tyr Glu Arg ArgVal Cys Phe 20 25 30 Leu Leu Leu Gln Leu Cys Asn Gly Leu Glu His Leu LysGlu His Gly 35 40 45 Ile Ile His Arg Asp Leu Cys Leu Glu Asn Leu Leu LeuVal His Cys 50 55 60 Thr Leu Gln Ala Gly Pro Gly Pro Ala 65 70 9 360 PRTHomo sapiens 9 Gly Gln Gly Trp Ser Trp Ala Gly Ile Pro Ser Ser Ala AlaAla Gln 1 5 10 15 Arg Ala Gly Pro Pro Ala Gly Ala Leu Glu Ala Leu SerPro Gly Gly 20 25 30 Ala Arg Ala His Ala Glu Arg Arg Gly Glu Met Arg AlaThr Pro Leu 35 40 45 Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys Lys Arg LeuGlu Leu Asp 50 55 60 Asp Asn Leu Asp Thr Glu Arg Pro Val Gln Lys Arg AlaArg Ser Gly 65 70 75 80 Pro Gln Pro Arg Leu Pro Pro Cys Leu Leu Pro LeuSer Pro Pro Thr 85 90 95 Ala Pro Asp Arg Ala Thr Ala Val Ala Thr Ala SerArg Leu Gly Pro 100 105 110 Tyr Val Leu Leu Glu Pro Glu Glu Gly Gly ArgAla Tyr Gln Ala Leu 115 120 125 His Cys Pro Thr Gly Thr Glu Tyr Thr CysLys Val Tyr Pro Val Gln 130 135 140 Glu Ala Leu Ala Val Leu Glu Pro TyrAla Arg Leu Pro Pro His Lys 145 150 155 160 His Val Ala Arg Pro Thr GluVal Leu Ala Gly Thr Gln Leu Leu Tyr 165 170 175 Ala Phe Phe Thr Arg ThrHis Gly Asp Met His Ser Leu Val Arg Ser 180 185 190 Arg His Arg Ile ProGlu Pro Glu Ala Ala Val Leu Phe Arg Gln Met 195 200 205 Ala Thr Ala LeuAla His Cys His Gln His Gly Leu Val Leu Arg Asp 210 215 220 Leu Lys LeuCys Arg Phe Val Phe Ala Asp Arg Glu Arg Lys Lys Leu 225 230 235 240 ValLeu Glu Asn Leu Glu Asp Ser Cys Val Leu Thr Gly Pro Asp Asp 245 250 255Ser Leu Trp Asp Lys His Ala Cys Pro Ala Tyr Val Gly Pro Glu Ile 260 265270 Leu Ser Ser Arg Ala Ser Tyr Ser Gly Lys Ala Ala Asp Val Trp Ser 275280 285 Leu Gly Val Ala Leu Phe Thr Met Leu Ala Gly His Tyr Pro Phe Gln290 295 300 Asp Ser Glu Pro Val Leu Leu Phe Gly Lys Ile Arg Arg Gly AlaTyr 305 310 315 320 Ala Leu Pro Ala Gly Leu Ser Ala Pro Ala Arg Cys LeuVal Arg Cys 325 330 335 Leu Leu Arg Arg Glu Pro Ala Glu Arg Leu Thr AlaThr Gly Ile Leu 340 345 350 Leu His Pro Trp Leu Arg Gln Asp 355 360 10146 PRT Homo sapiens UNSURE (140)..(140) UNSURE 10 Gln Arg Glu Ala AspMet His Arg Leu Phe Asn His Pro Asn Ile Leu 1 5 10 15 Arg Leu Val AlaTyr Cys Leu Arg Glu Arg Gly Ala Lys His Glu Ala 20 25 30 Trp Leu Leu LeuPro Phe Phe Lys Arg Gly Thr Leu Trp Asn Glu Ile 35 40 45 Glu Arg Leu LysAsp Lys Gly Asn Phe Leu Thr Glu Asp Gln Ile Leu 50 55 60 Trp Leu Leu LeuGly Ile Cys Arg Gly Leu Glu Ala Ile His Ala Lys 65 70 75 80 Gly Tyr AlaTyr Arg Asp Leu Lys Pro Thr Asn Ile Leu Leu Gly Asp 85 90 95 Glu Gly GlnPro Val Leu Met Asp Leu Gly Ser Met Asn Gln Ala Cys 100 105 110 Ile HisVal Glu Gly Ser Arg Gln Ala Leu Thr Leu Gln Asp Trp Ala 115 120 125 AlaGln Arg Cys Thr Ile Ser Tyr Arg Ala Pro Xaa Leu Phe Ser Val 130 135 140Gln Ser 145 11 505 PRT Homo sapiens 11 Met Leu Thr Ser Leu Asn Arg SerTrp Asn Glu Thr Thr Cys Cys Gly 1 5 10 15 Arg Ala Ser Phe Leu Glu LeuCys Thr Gly Gln Ile Gly Arg Thr Pro 20 25 30 Leu Gly Arg Arg Glu Gly MetGlu Asn Leu Lys His Ile Ile Thr Leu 35 40 45 Gly Gln Val Ile His Lys ArgCys Glu Glu Met Lys Tyr Cys Lys Lys 50 55 60 Gln Cys Arg Arg Leu Gly HisArg Val Leu Gly Leu Ile Lys Pro Leu 65 70 75 80 Glu Met Leu Gln Asp GlnGly Lys Arg Ser Val Pro Ser Glu Lys Leu 85 90 95 Thr Thr Ala Met Asn ArgPhe Lys Ala Ala Leu Glu Glu Ala Asn Gly 100 105 110 Glu Ile Glu Lys PheSer Asn Arg Ser Asn Ile Cys Arg Phe Leu Thr 115 120 125 Ala Ser Gln AspLys Ile Leu Phe Lys Asp Val Asn Arg Lys Leu Ser 130 135 140 Asp Val TrpLys Glu Leu Ser Leu Leu Leu Gln Val Glu Gln Arg Met 145 150 155 160 ProVal Ser Pro Ile Ser Gln Gly Ala Ser Trp Ala Gln Glu Asp Gln 165 170 175Gln Asp Ala Asp Glu Asp Arg Arg Ala Phe Gln Met Leu Arg Arg Asp 180 185190 Asn Glu Lys Ile Glu Ala Ser Leu Arg Arg Leu Glu Ile Asn Met Lys 195200 205 Glu Ile Lys Glu Thr Leu Arg Gln Tyr Leu Pro Pro Lys Cys Met Gln210 215 220 Glu Ile Pro Gln Glu Gln Ile Lys Glu Ile Lys Lys Glu Gln LeuSer 225 230 235 240 Gly Ser Pro Trp Ile Leu Leu Arg Glu Asn Glu Val SerThr Leu Tyr 245 250 255 Lys Gly Glu Tyr His Arg Ala Pro Val Ala Ile LysVal Phe Lys Lys 260 265 270 Leu Gln Ala Gly Ser Ile Ala Ile Val Arg GlnThr Phe Asn Lys Glu 275 280 285 Ile Lys Thr Met Lys Lys Phe Glu Ser ProAsn Ile Leu Arg Ile Phe 290 295 300 Gly Ile Cys Ile Asp Glu Thr Val ThrPro Pro Gln Phe Ser Ile Val 305 310 315 320 Met Glu Tyr Cys Glu Leu GlyThr Leu Arg Glu Leu Leu Asp Arg Glu 325 330 335 Lys Asp Leu Thr Leu GlyLys Arg Met Val Leu Val Leu Gly Ala Ala 340 345 350 Arg Gly Leu Tyr ArgLeu His His Ser Glu Ala Pro Glu Leu His Gly 355 360 365 Lys Ile Arg SerSer Asn Phe Leu Val Thr Gln Gly Tyr Gln Val Lys 370 375 380 Leu Ala GlyPhe Glu Leu Arg Lys Thr Gln Thr Ser Met Ser Leu Gly 385 390 395 400 ThrThr Arg Glu Lys Thr Asp Arg Val Lys Ser Thr Ala Tyr Leu Ser 405 410 415Pro Gln Glu Leu Glu Asp Val Phe Tyr Gln Tyr Asp Val Lys Ser Glu 420 425430 Ile Tyr Ser Phe Gly Ile Val Leu Trp Glu Ile Ala Thr Gly Asp Ile 435440 445 Pro Phe Gln Gly Cys Asn Ser Glu Lys Ile Arg Lys Leu Val Ala Val450 455 460 Lys Arg Gln Gln Glu Pro Leu Gly Glu Asp Cys Pro Ser Glu LeuArg 465 470 475 480 Glu Ile Ile Asp Glu Cys Arg Ala Ala Gly Arg Leu ValPro Arg Ser 485 490 495 Val Ala Ala Ala Arg Ala Val Asp Val 500 505 12499 PRT Homo sapiens 12 Phe Pro Leu Asp Val Glu Tyr Gly Gly Pro Asp ArgArg Cys Pro Pro 1 5 10 15 Pro Pro Tyr Pro Lys His Leu Leu Leu Arg SerLys Ser Glu Gln Tyr 20 25 30 Asp Leu Asp Ser Leu Cys Ala Gly Met Glu GlnSer Leu Arg Ala Gly 35 40 45 Pro Asn Glu Pro Glu Gly Gly Asp Lys Ser ArgLys Ser Ala Lys Gly 50 55 60 Asp Lys Gly Gly Lys Asp Lys Lys Gln Ile GlnThr Ser Pro Val Pro 65 70 75 80 Val Arg Lys Asn Ser Arg Asp Glu Glu LysArg Glu Ser Arg Ile Lys 85 90 95 Ser Tyr Ser Pro Tyr Ala Phe Lys Phe PheMet Glu Gln His Val Glu 100 105 110 Asn Val Ile Lys Thr Tyr Gln Gln LysVal Asn Arg Arg Leu Gln Leu 115 120 125 Glu Gln Glu Met Ala Lys Ala GlyLeu Cys Glu Ala Glu Gln Glu Gln 130 135 140 Met Arg Lys Ile Leu Tyr GlnLys Glu Ser Asn Tyr Asn Arg Leu Lys 145 150 155 160 Arg Ala Lys Met AspLys Ser Met Phe Val Lys Ile Lys Thr Leu Gly 165 170 175 Ile Gly Ala PheGly Glu Val Cys Leu Ala Cys Lys Val Asp Thr His 180 185 190 Ala Leu TyrAla Met Lys Thr Leu Arg Lys Lys Asp Val Leu Asn Arg 195 200 205 Asn GlnVal Ala His Val Lys Ala Glu Arg Asp Ile Leu Ala Glu Ala 210 215 220 AspAsn Glu Trp Val Val Lys Leu Tyr Tyr Ser Phe Gln Asp Lys Asp 225 230 235240 Ser Leu Tyr Phe Val Met Asp Tyr Ile Pro Gly Gly Asp Met Met Ser 245250 255 Leu Leu Ile Arg Met Glu Val Phe Pro Glu His Leu Ala Arg Phe Tyr260 265 270 Ile Ala Glu Leu Thr Leu Ala Ile Glu Ser Val His Lys Met GlyPhe 275 280 285 Ile His Arg Asp Ile Lys Pro Asp Asn Ile Leu Ile Asp LeuAsp Gly 290 295 300 His Ile Lys Leu Thr Asp Phe Gly Leu Cys Thr Gly PheArg Trp Thr 305 310 315 320 His Asn Ser Lys Tyr Tyr Gln Lys Gly Ser HisVal Arg Gln Asp Ser 325 330 335 Met Glu Pro Ser Asp Leu Trp Asp Asp ValSer Asn Cys Arg Cys Gly 340 345 350 Asp Arg Leu Lys Thr Leu Glu Gln ArgAla Arg Lys Gln His Gln Arg 355 360 365 Cys Leu Ala His Ser Leu Val GlyThr Pro Asn Tyr Ile Ala Pro Glu 370 375 380 Val Leu Leu Arg Lys Gly TyrThr Gln Leu Cys Asp Trp Trp Ser Val 385 390 395 400 Gly Val Ile Leu PheGlu Met Leu Val Gly Gln Pro Pro Phe Leu Ala 405 410 415 Pro Thr Pro ThrGlu Thr Gln Leu Lys Val Ile Asn Trp Glu Asn Thr 420 425 430 Leu His IlePro Ala Gln Val Lys Leu Ser Pro Glu Ala Arg Asp Leu 435 440 445 Ile ThrLys Leu Cys Cys Ser Ala Asp His Arg Leu Gly Arg Asn Gly 450 455 460 AlaAsp Asp Leu Lys Ala His Pro Phe Phe Ser Ala Ile Asp Phe Ser 465 470 475480 Ser Asp Ile Arg Lys His Pro Ala Pro Tyr Val Pro Thr Ile Ser His 485490 495 Pro Met Glu 13 375 DNA Homo sapiens 13 cttgcaggat ttgagttgaggaaaacacag acttccatga gtttgggaac tacgagagaa 60 aagacagaca gagtcaaatctacagcatat ctctcacctc aggaactgga agatgtattt 120 tatcaatatg atgtaaagtctgaaatatac agctttggaa tcgtcctctg ggaaatcgcc 180 actggagata tcccgtttcaaggctgtaat tctgagaaga tccgcaagct ggtggctgtg 240 aagcggcagc aggagccactgggtgaagac tgcccttcag agctgcggga gatcattgat 300 gagtgccggg cccatgatccctctgtgcgg ccctctgtgg atgaaatctt aaagaaactc 360 tccacctttt ctaag 375 14125 PRT Homo sapiens 14 Leu Ala Gly Phe Glu Leu Arg Lys Thr Gln Thr SerMet Ser Leu Gly 1 5 10 15 Thr Thr Arg Glu Lys Thr Asp Arg Val Lys SerThr Ala Tyr Leu Ser 20 25 30 Pro Gln Glu Leu Glu Asp Val Phe Tyr Gln TyrAsp Val Lys Ser Glu 35 40 45 Ile Tyr Ser Phe Gly Ile Val Leu Trp Glu IleAla Thr Gly Asp Ile 50 55 60 Pro Phe Gln Gly Cys Asn Ser Glu Lys Ile ArgLys Leu Val Ala Val 65 70 75 80 Lys Arg Gln Gln Glu Pro Leu Gly Glu AspCys Pro Ser Glu Leu Arg 85 90 95 Glu Ile Ile Asp Glu Cys Arg Ala His AspPro Ser Val Arg Pro Ser 100 105 110 Val Asp Glu Ile Leu Lys Lys Leu SerThr Phe Ser Lys 115 120 125 15 1961 DNA Homo sapiens 15 tcccgctggacgtggagtac ggaggcccag accggaggtg cccgcctccg ccctacccga 60 agcacctgctgctgcgcagc aagtcggagc agtacgacct ggacagcctg tgcgcaggca 120 tggagcagagcctccgtgcg ggccccaacg agcccgaggg cggcgacaag agccgcaaaa 180 gcgccaagggggacaaaggc ggaaaggata aaaagcagat tcagacctct cccgttcccg 240 tccgcaaaaacagcagagac gaagagaaga gagagtcacg catcaagagc tactcgccat 300 acgcctttaagttcttcatg gagcagcacg tggagaatgt catcaaaacc taccagcaga 360 aggttaaccggaggctgcag ctggagcaag aaatggccaa agctggactc tgtgaagctg 420 agcaggagcagatgcggaag atcctctacc agaaagagtc taattacaac aggttaaaga 480 gggccaagatggacaagtct atgtttgtca agatcaaaac cctggggatc ggtgcctttg 540 gagaagtgtgccttgcttgt aaggtggaca ctcacgccct gtacgccatg aagaccctaa 600 ggaaaaaggatgtcctgaac cggaatcagg tggcccacgt caaggccgag agggacatcc 660 tggccgaggcagacaatgag tgggtggtca aactctacta ctccttccaa gacaaagaca 720 gcctgtactttgtgatggac tacatccctg gtggggacat gatgagcctg ctgatccgga 780 tggaggtcttccctgagcac ctggcccggt tctacatcgc agagctgact ttggccattg 840 agagtgtccacaagatgggc ttcatccacc gagacatcaa gcctgataac attttgatag 900 atctggatggtcacattaaa ctcacagatt tcggcctctg cactgggttc aggtggactc 960 acaattccaaatattaccag aaagggagcc atgtcagaca ggacagcatg gagcccagcg 1020 acctctgggatgatgtgtct aactgtcggt gtggggacag gctgaagacc ctagagcaga 1080 gggcgcggaagcagcaccag aggtgcctgg cacattcact ggtggggact ccaaactaca 1140 tcgcacccgaggtgctcctc cgcaaagggt acactcaact ctgtgactgg tggagtgttg 1200 gagtgattctcttcgagatg ctggtggggc agccgccctt tttggcacct actcccacag 1260 aaacccagctgaaggtgatc aactgggaga acacgctcca cattccagcc caggtgaagc 1320 tgagccctgaggccagggac ctcatcacca agctgtgctg ctccgcagac caccgcctgg 1380 ggcggaatggggccgatgac ctgaaggccc accccttctt cagcgccatt gacttctcca 1440 gtgacatccggaagcatcca gccccctacg ttcccaccat cagccacccc atggacacct 1500 cgaatttcgaccccgtagat gaagaaagcc cttggaacga tgccagcgaa ggtagcacca 1560 aggcctgggacacactcacc tcgcccaata acaagcatcc tgagcacgca ttttacgaat 1620 tcaccttccgaaggttcttt gatgacaatg gctacccctt tcgatgccca aagccttcag 1680 gagcagaagcttcacaggct gagagctcag atttagaaag ctctgatctg gtggatcaga 1740 ctgaaggctgccagcctgtg tacgtgtaga tgggggccag gcacccccac cactcgctgc 1800 ctcccaggtcagggtcccgg agccggtgcc ctcacaggcc aatagggaag ccgagggctg 1860 ttttgttttaaattagtccg tcgattactt cacttgaaat tctgctcttc accaagaaaa 1920 cccaaacaggacacttttga aaacagcggt gccgcgaatt c 1961 16 588 PRT Homo sapiens 16 ProLeu Asp Val Glu Tyr Gly Gly Pro Asp Arg Arg Cys Pro Pro Pro 1 5 10 15Pro Tyr Pro Lys His Leu Leu Leu Arg Ser Lys Ser Glu Gln Tyr Asp 20 25 30Leu Asp Ser Leu Cys Ala Gly Met Glu Gln Ser Leu Arg Ala Gly Pro 35 40 45Asn Glu Pro Glu Gly Gly Asp Lys Ser Arg Lys Ser Ala Lys Gly Asp 50 55 60Lys Gly Gly Lys Asp Lys Lys Gln Ile Gln Thr Ser Pro Val Pro Val 65 70 7580 Arg Lys Asn Ser Arg Asp Glu Glu Lys Arg Glu Ser Arg Ile Lys Ser 85 9095 Tyr Ser Pro Tyr Ala Phe Lys Phe Phe Met Glu Gln His Val Glu Asn 100105 110 Val Ile Lys Thr Tyr Gln Gln Lys Val Asn Arg Arg Leu Gln Leu Glu115 120 125 Gln Glu Met Ala Lys Ala Gly Leu Cys Glu Ala Glu Gln Glu GlnMet 130 135 140 Arg Lys Ile Leu Tyr Gln Lys Glu Ser Asn Tyr Asn Arg LeuLys Arg 145 150 155 160 Ala Lys Met Asp Lys Ser Met Phe Val Lys Ile LysThr Leu Gly Ile 165 170 175 Gly Ala Phe Gly Glu Val Cys Leu Ala Cys LysVal Asp Thr His Ala 180 185 190 Leu Tyr Ala Met Lys Thr Leu Arg Lys LysAsp Val Leu Asn Arg Asn 195 200 205 Gln Val Ala His Val Lys Ala Glu ArgAsp Ile Leu Ala Glu Ala Asp 210 215 220 Asn Glu Trp Val Val Lys Leu TyrTyr Ser Phe Gln Asp Lys Asp Ser 225 230 235 240 Leu Tyr Phe Val Met AspTyr Ile Pro Gly Gly Asp Met Met Ser Leu 245 250 255 Leu Ile Arg Met GluVal Phe Pro Glu His Leu Ala Arg Phe Tyr Ile 260 265 270 Ala Glu Leu ThrLeu Ala Ile Glu Ser Val His Lys Met Gly Phe Ile 275 280 285 His Arg AspIle Lys Pro Asp Asn Ile Leu Ile Asp Leu Asp Gly His 290 295 300 Ile LysLeu Thr Asp Phe Gly Leu Cys Thr Gly Phe Arg Trp Thr His 305 310 315 320Asn Ser Lys Tyr Tyr Gln Lys Gly Ser His Val Arg Gln Asp Ser Met 325 330335 Glu Pro Ser Asp Leu Trp Asp Asp Val Ser Asn Cys Arg Cys Gly Asp 340345 350 Arg Leu Lys Thr Leu Glu Gln Arg Ala Arg Lys Gln His Gln Arg Cys355 360 365 Leu Ala His Ser Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro GluVal 370 375 380 Leu Leu Arg Lys Gly Tyr Thr Gln Leu Cys Asp Trp Trp SerVal Gly 385 390 395 400 Val Ile Leu Phe Glu Met Leu Val Gly Gln Pro ProPhe Leu Ala Pro 405 410 415 Thr Pro Thr Glu Thr Gln Leu Lys Val Ile AsnTrp Glu Asn Thr Leu 420 425 430 His Ile Pro Ala Gln Val Lys Leu Ser ProGlu Ala Arg Asp Leu Ile 435 440 445 Thr Lys Leu Cys Cys Ser Ala Asp HisArg Leu Gly Arg Asn Gly Ala 450 455 460 Asp Asp Leu Lys Ala His Pro PhePhe Ser Ala Ile Asp Phe Ser Ser 465 470 475 480 Asp Ile Arg Lys His ProAla Pro Tyr Val Pro Thr Ile Ser His Pro 485 490 495 Met Asp Thr Ser AsnPhe Asp Pro Val Asp Glu Glu Ser Pro Trp Asn 500 505 510 Asp Ala Ser GluGly Ser Thr Lys Ala Trp Asp Thr Leu Thr Ser Pro 515 520 525 Asn Asn LysHis Pro Glu His Ala Phe Tyr Glu Phe Thr Phe Arg Arg 530 535 540 Phe PheAsp Asp Asn Gly Tyr Pro Phe Arg Cys Pro Lys Pro Ser Gly 545 550 555 560Ala Glu Ala Ser Gln Ala Glu Ser Ser Asp Leu Glu Ser Ser Asp Leu 565 570575 Val Asp Gln Thr Glu Gly Cys Gln Pro Val Tyr Val 580 585

What is claimed is:
 1. An isolated polynucleotide molecule comprisingthe sequence of SEQ ID NO:5 or of SEQ ID NO:13.
 2. An isolatedpolynucleotide molecule encoding an amino acid sequence comprising thesequence of SEQ ID NO:11, or of SEQ ID NO:14, or of Leu-2 throughVal-505 of SEQ ID NO:11.
 3. A recombinant vector that directs theexpression of the polynucleotide molecule of claim
 2. 4. An isolatedpolypeptide having kinase activity encoded by the polynucleotidemolecule of claim
 1. 5. An isolated polypeptide according to claim 4having a molecular weight of approximately 58,001 Daltons as determinedby SDS-PAGE.
 6. An isolated polypeptide according to claim 4 innon-glycosylated form.
 7. A recombinant host cell comprising thepolynucleotide of claim
 2. 8. A method for the production of apolypeptide encoded by the polynucleotide of claim 2 comprisingculturing a recombinant host cell comprising the polynucleotide of claim2 under conditions promoting expression of said polypeptide.
 9. Themethod of claim 8, wherein the host cell is selected from the groupconsisting of bacterial cells, yeast cells, plant cells, insect cells,and animal cells.
 10. An isolated polypeptide having kinase activityproduced by the method of claim
 8. 11. An isolated polypeptide havingkinase activity comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:11, SEQ ID NO:14, and the amino acidsequence of Leu-2 through Val-505 of SEQ ID NO:11.
 12. The polypeptideof claim 11 comprising the amino acid sequence of SEQ ID NO:14.
 13. Thepolypeptide of claim 11 comprising the amino acid sequence of Leu-2through Val-505 of SEQ ID NO:11.
 14. An isolated polynucleotide moleculecomprising the sequence of SEQ ID NO:6 or of SEQ ID NO:15.
 15. Anisolated polynucleotide molecule encoding an amino acid sequencecomprising the sequence of SEQ ID NO:12 or of Pro-2 through Glu-499 ofSEQ ID NO:12.
 16. A recombinant vector that directs the expression ofthe polynucleotide molecule of claim
 15. 17. An isolated polypeptidehaving kinase activity encoded by the polynucleotide molecule of claim14.
 18. An isolated polypeptide according to claim 17 having a molecularweight of approximately 57,381 or 67,331 Daltons as determined bySDS-PAGE.
 19. An isolated polypeptide according to claim 17 innon-glycosylated form.
 20. A recombinant host cell comprising thepolynucleotide of claim
 15. 21. A method for the production of apolypeptide encoded by the polynucleotide of claim 15 comprisingculturing a recombinant host cell comprising the polynucleotide of claim15 under conditions promoting expression of said polypeptide.
 22. Themethod of claim 21, wherein the host cell is selected from the groupconsisting of bacterial cells, yeast cells, plant cells, insect cells,and animal cells.
 23. An isolated polypeptide having kinase activityproduced by the method of claim
 21. 24. An isolated polypeptide havingkinase activity comprising the amino acid sequence of Pro-2 throughGlu-499 of SEQ ID NO:12.